S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

Panagiotis S. Kabouridis; Anthony I. Magee; Steven C. Ley

doi:10.1093/emboj/16.16.4983

S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

Kabouridis, Panagiotis S.; Magee, Anthony I.; Ley, Steven C. 1997-08-15 00:00:00 The EMBO Journal Vol.16 No.16 pp.4983–4998, 1997 S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes 1,2 tion by interacting sequentially with two different families Panagiotis S.Kabouridis , 2,3 1,3 of cytoplasmic non-receptor PTKs. Anthony I.Magee and Steven C.Ley The ﬁrst of these is the SRC family, in particular LCK 1 2 Divisions of Cellular Immunology and Membrane Biology, and FYN (Perlmutter et al., 1993). Analysis of LCK- National Institute for Medical Research, The Ridgeway, Mill Hill, deﬁcient T-cell lines has indicated that LCK is absolutely London NW7 1AA, UK required for TCR signalling (Karnitz et al., 1992; Straus Corresponding authors and Weiss, 1992). Indeed, in the absence of LCK, the TCR fails to induce any tyrosine phosphorylation of LCK is a non-receptor protein tyrosine kinase required cytoplasmic proteins, and all downstream signalling events for signal transduction via the T-cell antigen receptor are blocked. Consistent with its important role in TCR (TCR). LCK N-terminus is S-acylated on Cys3 and signalling, T cell development is severely impaired in Cys5, in addition to its myristoylation on Gly2. Here LCK null mice (Molina et al., 1992). Genetic studies have the role of S-acylation in LCK function was examined. also indicated that FYN is involved in TCR signalling in Transient transfection of COS-18 cells, which express mature thymocytes (Appleby et al., 1992; Stein et al., a CD8-ζ chimera on their surface, revealed that LCK 1992) and, in certain T-cell lines, may be associated with mutants that were singly S-acylated were able to target the TCR (Samelson et al., 1990). However, from the to the plasma membrane and to phosphorylate CD8-ζ. phenotype of FYN null mice, it appears that the role of A non-S-acylated LCK mutant did not target to the FYN in TCR signalling may be restricted to particular plasma membrane and failed to phosphorylate CD8-ζ, stages of T-cell development (Appleby et al., 1992; Stein although it was catalytically active. Fusion of non-S- et al., 1992). acylated LCK to a transmembrane protein, CD16:7, The second family of PTKs which interact with the allowed its plasma membrane targeting and also TCR comprises ZAP-70 (Chan et al., 1992) and SYK phosphorylation of CD8-ζ when expressed in COS-18 (Taniguchi et al., 1991). After stimulation, ZAP-70 and cells. Thus S-acylation targets LCK to the plasma SYK are recruited to phosphorylated CD3 and ζ subunits membrane where it can interact with the TCR. When of the TCR, where they are in turn tyrosine phosphorylated expressed in LCK-negative JCam-1.6 T cells, delocal- (Chan et al., 1992, 1994b; Straus and Weiss, 1993; Wange ized, non-S-acylated LCK was completely non-func- et al., 1993; Chan and Shaw, 1996). ZAP-70 and SYK tional. Singly S-acylated LCK mutants, which were bind to the TCR via their two N-terminal SH2 domains expressed in part at the plasma membrane, efﬁciently which interact with speciﬁc phosphorylated tyrosines of reconstituted the induced association of phospho-ζ with the cytoplasmic tails of the CD3 complex (γδε) and ζ ZAP-70 and intracellular Ca ﬂuxes triggered by the dimers in immunoreceptor tyrosine-based activation motifs TCR. Induction of the late signalling proteins, CD69 (ITAMs) (Chan et al., 1994a; Chan and Shaw, 1996). and NFAT, was also reconstituted, although at reduced In an LCK-deﬁcient T cell line, TCR ITAMs are not levels. The transmembrane LCK chimera also sup- phosphorylated and ZAP-70 is not recruited to the TCR ported the induction of tyrosine phosphorylation and after stimulation, indicating that ZAP-70 plays a role Ca ﬂux by the TCR in JCam-1.6 cells. However, downstream of LCK (Straus and Weiss, 1992; Iwashima induction of ERK MAP kinase was reduced and the et al., 1994). Studies in COS cells have suggested that chimera was incapable of reconstituting induced CD69 one function of LCK is to phosphorylate ITAMs of the or NFAT expression. These data indicate that LCK TCR, thereby mediating association of the receptor with must be attached to the plasma membrane via dual ZAP-70 and its subsequent phosphorylation by LCK acylation of its N-terminus to function properly in (Iwashima et al., 1994; Chan et al., 1995) Consistent with TCR signalling. this hypothesis, both constitutive and inducible tyrosine Keywords: LCK/localization/S-acylation/signalling phosphorylation of ζ is abolished in LCK null thymocytes (van Oers et al., 1996). Tyrosine phosphorylation of ZAP- 70 activates its kinase activity (Chan et al., 1995; Wange et al., 1995) and, together with LCK, mediates tyrosine Introduction phosphorylation of multiple intracellular proteins which trigger T-cell proliferation. An essential role for ZAP-70 The induction of protein tyrosine kinase (PTK) activity in TCR signalling and T-cell development has been by the T-cell antigen receptor (TCR) is essential to couple it to downstream pathways which trigger proliferation and revealed by genetic studies in both murine and human differentiation of resting T cells into effector T cells systems (Hivroz and Fischer, 1994; Negishi et al., 1995). (Weiss and Littman, 1994). However, the component In contrast, SYK does not appear to be essential for αβ subunits of the TCR do not contain any intrinsic tyrosine T cell development (Turner et al., 1995), although the kinase domains. Rather, the TCR initiates signal transduc- development of epithelial γδ T cells is disrupted in SYK- © Oxford University Press 4983 P.S.Kabouridis, A.I.Magee and S.C.Ley Table I. Schematic representation of LCK acylation mutants Myr Pal Pal ↓↓ ↓ WT Met Gly Cys Val Cys Ser Ser Asn Pro Glu Asp C3A Met Gly Ala Val Cys Ser Ser Asn Pro Glu Asp C5A Met Gly Cys Val Ala Ser Ser Asn Pro Glu Asp C3,5A Met Gly Ala Val Ala Ser Ser Asn Pro Glu Asp The ﬁrst 11 amino acids of murine LCK are shown in three letter code, with amino acids that are modiﬁed by myristoylation (Myr) or S-acylation (Pal) in bold type. Mutated amino acids are shown in italics. negative mice (Mallick-Wood et al., 1996). The precise (C5A) or both cysteines (C3,5A) were substituted by an role of SYK in signalling via the TCR is presently unclear. alanine (Table I). The C3A, C5A and C3,5A mutants are However, recent experiments have indicated that SYK has myristoylated but they lack one (C3A and C5A) or both different activation requirements to ZAP-70 and does not (C3,5A) of the S-acyl attachment sites. require LCK expression (Chu et al., 1996). Thus it is Localization of the LCK mutants was ﬁrst analysed by likely that ZAP-70 and SYK play distinct roles in TCR transient expression in COS-18 cells and subcellular signalling. fractionation. Wild-type (WT) LCK was found exclusively Many proteins are now known to be post-translationally in the particulate fraction (Figure 1A). The majority of modiﬁed by the covalent addition of lipid moieties (Casey, the singly S-acylated C3A and C5A mutants, 71 and 92% 1995; Milligan et al., 1995). Prominent among these are respectively, was also found in the particulate fraction. In proteins involved in signalling via cell surface receptors, contrast, the minority of the C3,5A mutant was detected including the α and γ subunits of heterotrimeric G proteins in the particulate fraction (33%). This crude analysis (Milligan et al., 1995), small GTP-binding proteins such suggested that S-acylation of LCK was affecting its as Ras (Newman and Magee, 1993) and the SRC family localization. This was analysed in more detail by of protein tyrosine kinases (Resh, 1994). These lipid immunoﬂuorescence and confocal microscopy. In these modiﬁcations have been found to play key roles in experiments, transfected COS-18 cells were ﬁrst made association of these otherwise hydrophilic proteins with non-adherent and spherical by vigorous pipetting to facilit- the cytoplasmic face of speciﬁc cellular membranes. The ate detection of plasma membrane staining. WT LCK was N-terminal unique domain of LCK is modiﬁed by the detected exclusively at the plasma membrane, whereas addition of two different types of lipid (Shenoy-Scaria the C3,5A mutant was found diffusely throughout the et al., 1993; Koegl et al., 1994; Rodgers et al., 1994). cytoplasm and also in the nucleus (Figure 1C). The C3A Myristate, a saturated acyl group of 14 carbons, is added and C5A mutants showed an intermediate distribution and co-translationally to Gly2 via an amide bond, replacing were detected both at the plasma membrane and in the the initiator methionine (Johnson et al., 1994). Post- cytoplasm. translationally, two longer chain fatty acyl groups, which The role of acylation in LCK localization in T cells are often C16 palmitates, are attached by labile thioester was also investigated by stably expressing the panel of bonds to Cys3 and Cys5 (Milligan et al., 1995). In this mutants in a derivative of the Jurkat T-cell line, JCam- study, the role of S-acylation of LCK has been investigated 1.6, which expresses low levels of a catalytically inactive by analysis of point mutants expressed in COS-18 cells deletion mutant of LCK (Straus and Weiss, 1992). Sub- and in a leukaemic T-cell line which does not express cellular fractionation revealed that WT LCK was almost functional LCK. These experiments indicate that LCK exclusively in the particulate fraction (93%), similar to S-acylation is required for it to couple the TCR to COS-18 cells (Figure 1B). Removal of one or both downstream signalling pathways which, in part, reﬂects its S-acylation sites shifted the LCK into the soluble fraction. role in correct targeting of LCK to the plasma membrane. This shift was particularly pronounced with the C3A mutant where the majority of the protein (69%) was detected in the soluble fraction. Immunoﬂuorescence and Results confocal microscopy demonstrated that the WT protein N-terminal S-acylation is required for cortical was localized to the plasma membrane and to perinuclear targeting of LCK vesicles (Figure 1D), as shown previously (Ley et al., One of our laboratories has demonstrated previously that 1994a). The C3A and C5A mutants were also localized LCK is localized predominantly to the plasma membrane to the plasma membrane, although a signiﬁcant fraction and also to peri-centrosomal vesicles in human T lympho- was also detected diffusely distributed throughout the cytes (Ley et al., 1994a). The unique region of LCK is cytoplasm. In contrast, the C3,5A mutant was not detected modiﬁed by the addition of a myristate group to Gly2 at the plasma membrane but was present throughout the which is added co-translationally (Johnson et al., 1994). cytoplasm. Taken together, these data demonstrated that Recent studies from this and other laboratories have S-acylation was important in targeting LCK to the plasma indicated that the LCK unique region is also modiﬁed by membrane in both COS-18 and J-Cam-1.6 cells. the attachment of S-acyl groups to Cys3 and Cys5 (Shenoy- Scaria et al., 1993; Koegl et al., 1994; Rodgers et al., N-terminal S-acylation is not required for the 1994). To investigate the role of S-acylation of the LCK enzymatic activity of LCK unique region in its localization and function, three point Before analysing the signalling function of the LCK mutants were generated in which either Cys3 (C3A), Cys5 mutants, it was important to determine whether S-acylation 4984 S-acylation and signalling function of LCK Fig. 1. Subcellular localization of LCK mutants expressed in COS-18 and JCam-1.6 T cells. COS-18 cells (A and C) were transiently transfected with the LCK cDNA constructs indicated. JCam-1.6 cells (B and D) were stably transfected with plasmids encoding each of the panel of LCK mutants. Clones were then isolated in which the level of transfected LCK was similar or greater than transfected WT LCK. (A and B) Cells were disrupted by freeze–thawing and homogenization and then separated into soluble (S) and particulate (P) fractions by ultracentrifugation. Equal aliquots from the two fractions were then resolved by 10% SDS–PAGE, transferred onto PVDF membrane and probed with anti-LCK antibodies. The band that represents the transfected LCK is shown in brackets. In (B) the lower band represents the truncated, non-functional LCK form present in JCam-1.6 cells. (C and D) Cells were ﬁxed with paraformaldehyde, permeabilized and then stained for expression of LCK. The images shown are single confocal sections through representative transfected cells. Untransfected cells gave no detectable staining (data not shown). was required for LCK enzymatic activity. To investigate the mutants isolated from transfected J-Cam-1.6 T cells this, expression vectors encoding each mutant were trans- were found to have a speciﬁc activity similar to WT LCK fected into COS-18 cells which were then cultured for protein, as was found with transfected COS-18 cells 48 h. Total cell lysates were then resolved by SDS–PAGE (Figure 2C). and probed for phosphotyrosyl (PTyr) proteins. Figure 2A indicates that expression of each of the LCK mutants A non-S-acylated LCK mutant cannot resulted in induction of several PTyr proteins compared phosphorylate a CD8-ζ chimera expressed in with empty vector control. The most prominent of these COS-18 cells was a polypeptide of ~55 kDa which probably corres- COS-18 cells stably express on their surface a chimera ponded to transfected LCK itself. The C3,5A mutant, comprising the extracellular and transmembrane portions which was localized predominantly in the cytosol, was of the human CD8α chain fused to the cytoplasmic domain particularly active in the induction of PTyr proteins, many of the TCR ζ chain (Iwashima et al., 1994). Transient of which had mobilities distinct from those induced by expression of WT LCK in COS-18 cells results in phos- the plasma membrane-targeted LCK mutants. These data phorylation of the CD8-ζ chimera on tyrosines within indicated that there was no requirement for S-acylation ITAMs of the cytoplasmic tail of ζ. Co-expression of for LCK enzymatic activity in vivo. In addition, these ZAP-70 with LCK results in association of ZAP-70 with results suggested that the delocalized C3,5A LCK mutant phosphorylated ITAMs and its tyrosine phosphorylation. was accessible to distinct substrates from both the WT COS-18 cells, therefore, provide a simpliﬁed heterologous protein and singly S-acylated mutants that were associated model to study sequential interactions between the TCR with the plasma membrane. To assay the activity of LCK and the two non-receptor PTKs, LCK and ZAP-70. directly, the various mutants were immunoprecipitated As a ﬁrst step to investigate the role of LCK S-acylation from transfected COS-18 cells and tested for their ability in its functional interaction with the TCR, COS-18 cells to phosphorylate RR-SRC peptide in vitro. The RR-SRC were transfected with cDNAs encoding the various LCK peptide corresponds to residues 111–122 of SRC and mutants with or without ZAP-70 cDNA. As shown in contains its auto-phosphorylation site (Wong and Figure 3A (left side), WT LCK and singly S-acylated Goldberg, 1983). As shown in Figure 2B, all the C3A and C5A mutants were able to phosphorylate the S-acylation mutants had enzymatic activity comparable CD8-ζ chimera. In contrast, the C3,5A mutant was unable with the WT molecule. to increase the tyrosine phosphorylation of the chimera The LCK mutants were also immunoprecipitated from above background levels although this mutant was the panel of stably transfected JCam-1.6 cell lines and expressed at levels comparable with WT LCK (Figure assayed for their activity in vitro. To increase sensitivity 3B). ZAP-70 associated with the CD8-ζ chimera, resulting in these assays, a different peptide, referred to as C-peptide, in its increased tyrosine phosphorylation, when co- was used, which has been demonstrated to be the optimal expressed with WT LCK or either of the two singly substrate for LCK (Songyang and Cantley, 1995). All of S-acylated mutants (Figure 3A, right side). The non- 4985 P.S.Kabouridis, A.I.Magee and S.C.Ley Fig. 3. Induction of CD8-ζ phosphorylation by LCK mutants in COS-18 cells. COS-18 cells were transiently transfected with vectors encoding the various LCK mutants in the presence and absence of ZAP-70 cDNA. (A) CD8-ζ was immunoprecipitated from cell lysates using the OKT8 mAb, resolved by 10% SDS–PAGE and transferred electrophoretically to PVDF membrane. The blot was then probed sequentially with an anti-PTyr mAb (PY) followed by an anti-ζ antibody (zeta). The positions of phospho-ZAP-70 (ZAP-70-PO ) and phospho-CD8-ζ (CD8-ζ-PO ) are indicated. H denotes the heavy chain of the immunoprecipitating antibody. (B) Equivalent amounts of total cell lysates from the transfected COS-18 cells used in (A) were resolved by SDS–PAGE. The expression of transfected LCK and ZAP-70 was then checked by sequential immunoblotting of the PVDF membrane with the appropriate antibodies. S-acylated C3,5A mutant, however, was again unable to induce phosphorylation of CD8-ζ or its association with ZAP-70 in the doubly transfected cells. Western blot analysis demonstrated that this mutant and the co-trans- fected ZAP-70 were expressed at levels similar to that achieved in the WT LCK transfection (Figure 3B). In conclusion, these experiments indicated that non-S- acylated LCK, which did not stably interact with the Fig. 2. LCK S-acylation mutants are enzymatically active both in vivo plasma membrane, also failed to phosphorylate CD8-ζ and in vitro.(A) Lysates were prepared from COS-18 cells transiently when expressed in COS-18 cells. transfected with plasmids encoding the various LCK acylation mutants, as indicated. Proteins were resolved by SDS–PAGE, transferred onto PVDF membrane and immunoblotted with anti-PTyr Non-S-acylated LCK cannot reconstitute early mAb. The PVDF membrane was then stripped and reprobed with signalling events triggered by the TCR in JCam-1.6 anti-LCK antiserum to conﬁrm expression of the transfected LCK T cells protein (not shown). (B) LCK mutants from transiently transfected JCam-1.6 is a mutant derivative of the Jurkat T-cell line COS-18 cells were immunoprecipitated and assayed for their ability to phosphorylate RR-SRC peptide in vitro. Results shown are normalized which expresses low levels of a mutant form of LCK against the amount of LCK in immunoprecpitates as determined by that is catalytically inactive (Straus and Weiss, 1992). immunoblotting, and are expressed in arbitrary units. Data are the Stimulation of JCam-1.6 T cells with CD3 antibody fails mean ( SE) of duplicate assays. (C) LCK was immunoprecipitated to induce early or late signalling events triggered by the from JCam-1.6 clones expressing the indicated LCK mutants and assayed for its ability to phosphorylate C-peptide. Results are TCR. TCR signalling, however, may be reconstituted by presented as in (B). transfection of JCam-1.6 cells with WT LCK. To investi- 4986 S-acylation and signalling function of LCK Fig. 4. Analysis of early signalling events triggered by the TCR in LCK-transfected JCam-1.6 cells. (A) Each of the clones was stimulated for 5 min with 1 μg/ml of F(ab) fragments of the CD3 antibody OKT3 or left unstimulated and then lysed in 1% NP-40 lysis buffer. ZAP-70 was then isolated from cell lysates by immunoprecipitation and resolved by 12.5% SDS–PAGE. Tyrosine-phosphorylated proteins were detected by immunoblotting with an anti-PTyr mAb. The positions of phospho-ZAP-70 and phospho-ζ are indicated on the left of the panel. To check that equivalent levels of ZAP-70 were present in each immunoprecipitate, the PVDF membrane was stripped and probed with an anti-ZAP-70 antibody. Qualitatively similar results were obtained with at least two other clones for each of the LCK mutants (data not shown). (B) Transfected JCam-1.6 cells were loaded with the Ca -interacting agent Indo-1 and then stimulated with OKT3 F(ab) antibody. Changes in the the level of intracellular free Ca are shown as a function of time. Successful loading with Indo-1 was conﬁrmed by subsequently treating the cells with ionomycin. The times at which CD3 antibody and ionomycin were added are indicated. gate the role of LCK acylation in signalling via the TCR although several bands were constitutively phosphorylated in T cells, JCam-1.6 cells were transfected with various when compared with the empty vector control (data not LCK mutants and stable clones expressing levels of shown). Thus, S-acylation of LCK was essential for the transfected LCK similar to or greater than that of WT TCR to induce tyrosine phosphorylation of any intra- LCK were selected for analysis. For each LCK mutant, cellular proteins. qualitatively similar results to those shown below were The panel of JCam-1.6 clones was also analysed for obtained with other clones tested (data not shown). their ability to induce increases in intracellular free Ca As discussed in the Introduction, TCR cross-linking following TCR cross-linking. Both singly S-acylated LCK rapidly induces LCK to phosphorylate ITAMs of the ζ mutants induced Ca levels similar to that achieved in chain (Weiss and Littman, 1994). ZAP-70 is then recruited the WT clone (Figure 4B). However, non-S-acylated to the TCR via binding of its two N-terminal SH2 domains C3,5A mutant failed to induce any increase in intracellular and is itself tyrosine phosphorylated. To investigate the free Ca following TCR cross-linking. Taken together, importance of LCK S-acylation in these early signalling these data indicated that the LCK S-acylation mutants events, the various JCam-1.6 clones were stimulated which were at least in part targeted to the plasma membrane with F(ab) fragments of the mitogenic CD3 monoclonal were able to reconstitute early signalling events triggered antibody OKT3 for 5 min and ZAP-70 protein was by the TCR. In contrast, the non-S-acylated C3,5A mutant, immunoprecipitated from cell lysates. Introduction of WT which was completely delocalized, was non-functional. LCK or either of the singly S-acylated mutants into JCam- 1.6 reconstituted the ability of the TCR to induce tyrosine LCK S-acylation mutants are defective in phosphorylation of ZAP-70 and its association with the reconstituting late signalling events triggered by TCR (Figure 4A). In contrast, non-S-acylated C3,5A LCK the TCR in JCam-1.6 cells failed to reconstitute inducible phosphorylation of ZAP- Late activation events following stimulation of the TCR 70 or its association with phospho-ζ, although this clone include induction of the cell surface activation antigen, expressed high levels of transfected LCK. CD69 (Testi et al., 1989), and of the T cell-speciﬁc Similarly to the results with ZAP-70 tyrosine phos- transcription factor, NFAT, which is involved in transcrip- phorylation, TCR cross-linking of cell lines expressing tional regulation of the interleukin-2 (IL-2) gene (Jain singly acylated LCK induced qualitatively similar patterns et al., 1995). In order to investigate the ability of the of total PTyr proteins to the WT LCK transfectant (data various LCK mutants to support the ﬁrst of these late not shown). In contrast, the C3,5A LCK mutant failed activation events in JCam-1.6 cells, the panel of clones to support any TCR-induced tyrosine phosphorylation, was stimulated with CD3 antibody for 48 h, following 4987 P.S.Kabouridis, A.I.Magee and S.C.Ley mutant was slightly impaired relative to WT in the induction of NFAT, whereas the C3A mutant induced only 30% of WT levels of NFAT following CD3 stimulation. The C3,5A mutant was essentially inactive. All transfected cell lines induced similar levels of NFAT expression in response to stimulation with Ca ionophore and phorbol ester (data not shown). In conclusion, the experiments in this section indicate that both singly S-acylated LCK mutants were able to reconstitute late signalling pathways triggered by the TCR. However, neither of these mutants was as effective as the WT LCK protein at inducing these late signalling events. This suggested that all three attached lipid groups were required for LCK to work with maximum efﬁciency in reconstituting TCR signalling in the JCam-1.6 cells. The non-S-acylated C3,5A mutant was not capable of coupling the TCR to late signalling events, as expected from its inability to induce early signalling events following TCR cross-linking. Retargeting of non-S-acylated LCK to the plasma membrane reconstitutes early TCR-induced signalling events in JCam-1.6 cells The experiments in both COS-18 cells and JCam-1.6 cells revealed a correlation between plasma membrane targeting of LCK mutants and their ability to interact functionally with the TCR. These results, therefore, suggested that one of the primary functions of LCK S-acylation is to target it to the plasma membrane. To investigate this hypothesis, Fig. 5. Analysis of late signalling events triggered by the TCR in LCK-transfected JCam-1.6 cells. (A) A total of 110 cells from each the function was tested of LCK which could be expressed of the JCam-1.6 clones were cultured in the presence of 10 μg/ml at the plasma membrane independently of its lipid modi- OKT3 F(ab) antibody for 48 h. CD69 expression was then ﬁcations. A chimera was constructed by fusing the entire determined by immunoﬂuorescence and ﬂow cytometry. The LCK coding sequence to the extracellular domain of percentage of cells which were CD69 positive was determined using an FITC-conjugated mouse myeloma to set the background levels of CD16 and the transmembrane domain of CD7 to generate staining. Similar results were obtained in two other experiments. 16:7:LCK-WT, as shown in Figure 6A (Kolanus et al., (B) LCK-expressing JCam-1.6 cells were transiently transfected with 1993; kindly provided by Brian Seed, Boston, MA). This 15 μg of the NFAT–luciferase reporter construct, pBR322-3NFAT- protein contains intact S-acylation sites on LCK but cannot Luc. The cells were then cultured for 2 h and stimulated with be myristoylated. A mutant of this chimera was generated 10 μg/ml OKT3 antibody () or left unstimulated (–). After a further 12 h in culture, cells were lysed and luciferase activity assayed. Data by PCR in which the two S-acylation sites on LCK were are presented in arbitrary units as a mean of duplicate measurements mutated to generate the 16:7:LCK-C3,5A chimera. In vivo ( SE). Qualitatively similar results were obtained in three separate labelling with [ H]palmitate conﬁrmed that the 16:7:LCK- experiments. C3,5A chimera was not S-acylated, in contrast to 16:7:LCK-WT (data not shown). The functional experi- which the cells were analysed for CD69 expression by ments described below were all carried out on the non- immunoﬂuorescence and ﬂow cytometry. WT LCK acylated 16:7:LCK-C3,5A chimera. Similar results were induced expression of CD69 as expected (Figure 5A). obtained with 16:7:LCK-WT chimera (data not shown). Both singly S-acylated mutants were able to induce the In initial experiments, cDNAs encoding WT LCK, LCK expression of CD69, although the level of induced expres- C3,5A and the 16:7:LCK-C3,5A chimera were transiently sion in C3A mutant-transfected cells was markedly expressed in COS-18 cells and tested for their ability to reduced compared with WT LCK. The C3,5A mutant was phosphorylate CD8-ζ. As shown previously, WT LCK, completely unable to support the induction of CD69 but not the C3,5A mutant, could phosphorylate CD8-ζ. expression following TCR stimulation. However, the 16:7:LCK-C3,5A chimera was able to induce In order to assay the induction of NFAT transcription high levels of tyrosine phosphorylation of CD8-ζ (Figure factor, each JCam-1.6 clone was transfected with a con- 6B). Thus retargeting to the plasma membrane overcame struct containing the luciferase reporter gene under the the acylation requirement for LCK to interact functionally control of three copies of the NFAT regulatory element with CD8-ζ when expressed in COS-18 cells. Co-transfec- (Verweij et al., 1990). After 2 h in culture, cells were tion of ZAP-70 cDNA with 16:7:LCK-C3,5A cDNA also stimulated with CD3 antibody, or left unstimulated, and resulted in the association of ZAP-70 with CD8-ζ and its recultured for a further 12 h before harvesting. Cell lysates tyrosine phosphorylation (data not shown). were then prepared and luciferase activity assayed. A To study the function of 16:7:LCK-C3,5A in T cells, similar pattern of responsiveness was seen as with CD69 this cDNA construct was stably transfected into JCam- expression. Thus WT LCK induced NFAT following CD3 1.6 cells and an oligoclonal population of cells isolated, cross-linking as expected. The singly S-acylated C5A using ﬂuorescence activated cell sorting (FACS), which 4988 S-acylation and signalling function of LCK Fig. 6. Analysis of 16:7:LCK-C3,5A LCK chimera expressed in COS-18 and JCam-1.6 cells. (A) Schematic depiction of the 16:7:LCK-C3,5A chimera used in this study. (B) COS-18 cells were transfected with the indicated expression vectors and then CD8-ζ immunoprecipitated from cell lysates and immunoblotted sequentially for PTyr and ζ. The position of phospho-CD8-ζ is indicated on the left of the panel. Expression of transfected LCK or 16.7:LCK chimera was conﬁrmed by immunoblotting of aliquots of total cell lysate used for immunoprecipitation, with an anti-LCK antibody (data not shown). (C) The expression level of the 16:7:C3,5A LCK chimera in stably transfected JCam-1.6 cells was determined by ﬂow cytometry (dashed line). The background ﬂuorescence was set with an FITC-conjugated anti-Ig antibody (solid line). Fig. 7. Analysis of early tyrosine phosphorylation events triggered by the 16:7:LCK-C3,5A chimera in transfected JCam-1.6 cells. (A) JCam-1.6 cells stably expressing on their surface the 16:7:LCK-C3,5A chimera were stimulated with CD3 and/or CD16 antibodies in the presence of anti-IgG antibody for 5 min or left unstimulated. Control JCam-1.6 cells transfected with WT or C3,5A LCK were stimulated with CD3 plus anti-Ig antibodies. ZAP-70 was then immunoprecipitated from cell lysates and immune complexes were resolved by 12.5% SDS–PAGE and immunoblotted with anti-PTyr mAb (upper panel). The positions of phospho-ZAP-70 (ZAP-70-PO ) and phospho-ζ (ζ-PO ) are indicated on the left of the panels. 4 4 Blots were re-probed with anti-ZAP-70 antibody to conﬁrm that equivalent amounts of antigen were immunoprecipitated from each of the JCam-1.6 clones (lower panel). (B) A total of 1010 cells of the WT- or 16:7:LCK-C3,5A-expressing JCam-1.6 cells were left untreated or were stimulated as in (A). Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with the anti-PTyr mAb 4G10, resolved by SDS–PAGE electrophoresis and detected with Western blotting using anti-PTyr mAb. The asterisk indicates the migration distance of the 16:7: LCK-C3,5A chimera. (C) LCK WT- and 16:7:LCK-C3,5A-expressing JCam-1.6 cells were stimulated as in (A) for the indicated times, and cell lysates representing 0.510 cells were analysed by 10% SDS–PAGE and probed with anti-phosphotyrosine antibodies. The asterisk indicates the 16:7:LCK-C3,5A chimera while the arrow shows the activation-induced 36 kDa phosphoprotein. 4989 P.S.Kabouridis, A.I.Magee and S.C.Ley Fig. 9. Analysis of late signalling events triggered by the Fig. 8. Short and long term Ca ﬂuxes in WT- and 16:7:LCK-C3,5A- 16:7:LCK-C3,5A chimera in transfected JCam-1.6 cells. (A) A total of expressing cells. (A) The indicated transfected JCam-1.6 cells were 110 cells of each of the indicated JCam-1.6 clones were cultured loaded with Indo-1 and then sequentially stimulated with CD16 with CD3 antibody, CD16 antibody, or both, as indicated, plus anti- followed by CD3 and anti-IgG (goat anti-mouse) antibodies. IgG antibody for 48 h, or left unstimulated. The percentage of cells Fluctuations in the levels of intracellular free Ca before and after which expressed CD69 on their surface was then determined as in antibody stimulation are shown as a function of time. Successful Figure 5. (B) Transfected JCam-1.6 cells were transiently loading with Indo-1 was conﬁrmed by subsequently treating the cells transfected with 15 μg of the NFAT–luciferase reporter construct, with ionomycin (not shown). The times at which antibodies were pBR322-3NFAT-Luc. The cells were then cultured for2hand added are indicated. (B) The WT- and 16:7:C3,5A-expressing clones stimulated with CD3 antibody, CD16 antibody, or both in the presence were loaded with Indo-1 as in (A) and basal and stimulation-induced of anti-IgG antibody. After a further 12 h in culture, cells were lysed intracellular Ca levels were monitored in a FACS. For each time and luciferase activity assayed. Data are presented in arbitrary units as point, ~110 cells were analysed and the violet/blue emission ratio a mean of duplicate measurements ( SE). was determined. All the values collected were plotted as the emission ratio versus time. This is one of two experiments performed with similar results. 1.6 cells cells expressing WT LCK were stimulated with showed high levels of surface CD16 staining (Figure 6C). CD3 mAb and anti-Ig (Figure 8A). A kinetic experiment Stimulation of the TCR on cells expressing 16:7:LCK- also demonstrated that the LCK chimera was able to C3,5A failed to induce tyrosine phosphorylation of ZAP- sustain TCR-induced intracellular Ca levels above basal 70 or its association with the ζ chain (Figure 7A). In levels for upto 2 h post-stimulation, similarly to WT LCK contrast, WT LCK induced both of these events as (Figure 8B). These results, therefore, suggested that there expected. However, if the chimera was stimulated with was no requirement for S-acylation of the plasma mem- both CD3 and CD16 antibodies co-cross-linked with anti- brane retargeted LCK chimera to induce early signalling Ig, ZAP-70 phosphorylation and association with the ζ events triggered by the TCR. However, analysis of CD69 chain was strongly induced. Control experiments demon- and NFAT induction revealed that the chimera was unable strated that addition of CD3 and CD16 antibodies in to induce these late activation events, even when the TCR the absence of anti-Ig was insufﬁcient to reconstitute and CD16 were co-aggregated on the cell surface (Figure signalling, suggesting that the chimera had to be brought 9A and B). In contrast, the WT LCK-expressing clone into close proximity with the TCR in order to function was able to induce both of these events, as expected. (data not shown). The chimera was also able to reconstitute Thus the chimeric transmembrane form of LCK did not rapid increases in intracellular free Ca when the TCR reconstitute TCR coupling to late signalling events in was co-cross-linked with CD16. This increase consistently was found to be greater than that achieved when JCam- JCam-1.6 T cells. 4990 S-acylation and signalling function of LCK Fig. 10. Kinetics of ERK activation following stimulation of WT- and 16:7:LCK-C3,5A-expressing JCam-1.6 cells. WT- and 16:7:LCK-C3,5A- expressing JCam-1.6 cells were left untreated or were stimulated with OKT3 or OKT3 plus CD16 mAbs respectively, followed by cross-linking with anti-Ig antibody for the indicated times, and the phosphorylation of ERK-1 and ERK-2, in equal cell lysate aliquots, was detected by immunoblotting with anti-phospho-ERK antibody (upper panel). The same membrane was stripped and reprobed with anti-ERK-1/2 antiserum (lower panel). The migrating distances for ERK-1 and ERK-2 are indicated with arrows. The slower migrating species observed in the lower panel are indicative of ERK activation. The 16:7:LCK-C3,5A chimera is deﬁcient in its ability to reconstitute TCR-induced ERK activation in JCam-1.6 cells The failure of the 16:7:LCK-C3,5A chimera to reconstitute late signalling events triggered by the TCR was not due to a quantitative reduction in the level of phosphorylation of ZAP-70 or its association with phospho-ζ (Figure 7A). To investigate whether the chimera was able to reconstitute the tyrosine phosphorylation of other intracellular proteins after TCR cross-linking, JCam-1.6 cells transfected with WT LCK or 16:7:LCK-C3,5A were stimulated with the indicated antibodies and PTyr proteins immunoprecipitated and Western blotted with an anti-PTyr monoclonal anti- body (mAb). In Figure 7B, it can be seen that the pattern of PTyr bands induced by the TCR was very similar for JCam-1.6 cells transfected with either LCK construct. Thus the 16:7:LCK-C3,5A chimera appeared to facilitate the phosphorylation of all the major TCR-inducible PTyr Fig. 11. The LCK chimera can interact with ZAP-70 and, in 16:7:LCK-C3,5A-expressing cells, co-expression of WT but not C3,5A proteins. A time-course experiment also indicated that the LCK, can support TCR-induced NFAT production. Also the kinetics of TCR-induced phosphorylation of most of the submembrane topology of the 16:7:C3,5A chimera differs from that of major PTyr proteins in both cell lines were similar (Figure WT LCK. (A) Cells (3010 ) expressing WT or 16:7:C3,5A were 7C). However, the phosphorylation of a 36 kDa PTyr stimulated with OKT3 or OKT3/CD16 mAb respectively followed by protein (shown with an arrow) was found consistently to cross-linking with anti-mouse Ig, for 5 min. Following stimulation, ZAP-70 was precipitated from cell lysates using Afﬁgel-coupled be more transient in cells expressing the 16:7:LCK-C3,5A phospho-ITAM1 peptides. Immune complexes were resolved chimera compared with cells reconstituted with WT LCK. in 10% SDS–PAGE, transferred onto PVDF membrane, and Activation of the Ras-MAP kinase pathway is required co-immunoprecipitated LCK was detected with speciﬁc antibodies for induction of CD69 and NFAT by the TCR (D’Ambrosio (upper panel). The same membrane was stripped and reprobed with et al., 1994; Genot et al., 1996). To investigate the ZAP-70-reacting antibodies (lower panel). The experiment shown is representative of four experiments done with similar results. possibility that ERK MAP kinase activation might be (B) A total of 2010 16:7:LCK-C3,5A-expressing cells were affected in the 16:7:LCK-C3,5A chimera-transfected co-transfected with 15 μg of the NFAT–luciferase construct plus 25 μg JCam-1.6 cells, total cell lysates were prepared from cells of WT- or C3,5A LCK-containing plasmids as indicated. Activation of stimulated for the indicated times with anti-CD3 mAb and the cells and luciferase assays were performed as in Figure 9. The electroblotted. Blots were then probed with a phospho- experiment shown is one of four performed with identical results. Treatment of the transfected cells with ionophore and phorbol ester speciﬁc anti-ERK-1/2 antibody which recognizes the activ- induced comparable levels of NFAT-driven luciferase activity. ated, tyrosine-phosphorylated forms of ERKs 1 and 2. (C) JCam-1.6 cells (5010 ) expressing WT or 16:7:LCK-C3,5A were The blot was then stripped and reprobed with an anti- lysed in 1% Triton X-100-containing buffer and the cell lysates were ERK-1/2 antibody to detect the total amount of ERKs 1 fractionated through a sucrose gradient as described in Materials and and 2 in each lane. In Figure 10, it can be seen that methods. The content of LCK in equal aliquots from high (H) and low (L) density sucrose fractions was assessed by Western analysis. The activation of ERKs 1 and 2 was deﬁcient in the 16:7:LCK- blot was intentionally overexposed in order to detect even minimal C3,5A chimera-transfected JCam-1.6 cells compared with presence of LCK in the low density fraction. the WT LCK transfectant. These data raised the possibility that inability of the LCK chimera to reconstitute late It has been suggested that this interaction may be necessary signalling events triggered by the TCR might result from for the functional cooperation between these PTKs during its inability to support the efﬁcient activation of ERK TCR signalling (Duplay et al., 1994; Straus et al., 1996). MAP kinases. It was possible that the failure of 16:7:LCK-C3,5A to The 16:7:LCK-C3,5A chimera associates with reconstitute CD69 and NFAT expression induced by the ZAP-70 after TCR stimulation TCR in the JCam-1.6 cells might have resulted from its TCR stimulation induces LCK to associate with ZAP-70 inability to form a complex with ZAP-70. To investigate via the former protein’s SH2 domain (Duplay et al., 1994). this possibility, lysates were prepared from JCam-1.6 cells, 4991 P.S.Kabouridis, A.I.Magee and S.C.Ley expressing either WT LCK or 16:7:LCK-C3,5A, with and with GEMs (Shenoy-Scaria et al., 1993, 1994; Rodgers without mAb stimulation of their TCRs. ZAP-70 was then et al., 1994). GEMs also contain glycosylphosphatidinosi- isolated by incubating lysates with a synthetic phosphoryl- tol (GPI)-linked proteins (Mayor et al., 1994; Rodgers ated oligopeptide, corresponding to the membrane- et al., 1994) and heterotrimeric G proteins (Sargiacomo proximal ITAM of the ζ chain (Osman et al., 1995), et al., 1993). The association of LCK with both GPI- coupled to Afﬁ-Gel 10 beads (Bio-Rad). The phospho- anchored proteins and with GEMs is dependent on its ITAM peptide interacted with ZAP-70 via its SH2 domains S-acylation (Shenoy-Scaria et al., 1993; Rodgers et al., (Weiss and Littman, 1994), and, as a consequence, isolated 1994). Thus GEMs may be specialized microdomains that ZAP-70 was not bound to phospho-ITAMs of the TCR. are involved in coupling GPI-linked receptors to activation This method of puriﬁcation of ZAP-70, therefore, avoided of PTK activity. the possibility of isolating the 16:7:LCK-C3,5A chimera It was possible that the 16:7:LCK-C3,5A chimera, artefactually via its association with the TCR induced although it was targeted to the plasma membrane, might by the co-cross-linked CD3 and CD16 mAbs used for not be accessible to GEMs. To investigate this possibility, stimulating the cells. In Figure 11A, it can be seen that cell lysates were prepared from WT- and 16:7:LCK- both WT LCK and the 16:7:LCK-C3,5A chimera inducibly C3,5A-transfected JCam-1.6 cells. These lysates were associated with ZAP-70 after stimulation of the TCR. then resolved on a discontinuous sucrose gradient by Thus the failure of 16:7:LCK-C3,5A to fully complement centrifugation and low density (30/5% sucrose interface) TCR signalling in the JCam-1.6 did not result from its and high density (40% sucrose) fractions collected. GEMs failure to interact with ZAP-70. partition into the low density fraction (Brown and Rose, 1992). Fractionated lysates were then probed for LCK; as Expression of cytosolic C3,5A LCK does not expected WT LCK was clearly detected in the low density complement the signalling defect of the GEM fraction (Figure 11C). However, even after long 16:7:LCK-C3,5A chimera exposure, none of the 16:7:LCK-C3,5A chimera was The S-acyl moieties on LCK turn over with a half-life detected in the GEM fraction. These data, therefore, that is much shorter than the half-life of the protein (Paige suggested that, within the plane of the plasma membrane, et al., 1995). Deacylation may allow LCK to detach from the 16:7:LCK-C3,5A chimera was not targeted identically the membrane after activation (Milligan et al., 1995). to the WT LCK protein. Thus the failure of the 16:7:LCK-C3,5A chimera to support the induction of late activation events might have Discussion resulted from a requirement for detachment of LCK from the plasma membrane after TCR stimulation. This could This study demonstrates that S-acylation of the unique not occur when LCK was anchored artiﬁcially via a region of LCK is essential for its targeting to the plasma transmembrane domain, as was the case for 16:7:LCK- membrane. Myristoylation of LCK on its own was insufﬁ- C3,5A. cient to attach LCK ﬁrmly to the plasma membrane, as To investigate the possibility that LCK might need to revealed by analysis of the non-S-acylated C3,5A LCK detach from the plasma membrane to carry out its signall- mutant which was localized throughout the cytoplasm ing functions, 16:7:LCK-C3,5A-expressing cells were (Figure 1). This is consistent with experiments analysing transiently transfected with cDNAs encoding either WT the binding of myristoylated peptides to phospholipid or C3,5A LCK together with the NFAT luciferase reporter vesicles, which indicate that myristoylation of a protein construct. The cells were then stimulated with co-cross- cannot stably bind it to a lipid bilayer (Peitzsch and linked CD3 and CD16 mAbs, or left unstimulated, and McLaughlin, 1993; Bhatnagar and Gordon, 1997). To NFAT-driven luciferase production was assayed after 12 h achieve detectable binding of LCK to membranes, in culture. Transfection of WT LCK restored TCR induc- S-acylation was required in addition to myristoylation, as tion of NFAT in the 16:7:LCK-C3,5A-expressing JCam- indicated by the partial localization of singly S-acylated 1.6 cells. Thus the TCR signalling pathways leading to LCK to the plasma membrane. However, analysis of the NFAT production were intact in the JCam-1.6 cells which distribution of WT LCK indicated that the attachment of expressed 16:7:LCK-C3,5A. However, TCR induction of two S-acyl groups and a myristate group was necessary NFAT was not restored in cells transfected with cytosolic to achieve high levels of membrane binding. The difference C3,5A LCK (Figure 11B). These data, therefore, did not in localization of WT LCK and the two singly S-acylated suggest that the signalling defect of 16:7:LCK-C3,5A was mutants also suggests that most molecules of the WT due to its inability to detach from the plasma membrane protein carry three attached acyl groups. after TCR stimulation. In a separate series of experiments, this laboratory recently has demonstrated that the addition of the ﬁrst The 16:7:LCK-C3,5A chimera is excluded from 10 amino acids of LCK to two different soluble cyto- glycolipid-enriched microdomains plasmic proteins was sufﬁcient to retarget them to the The plasma membrane is specialized into microdomains plasma membrane and also to vesicles next to the nucleus which are enriched in glycosphingolipids, sphingomyelin in COS-7 cells (Zlatkine et al., 1997). Taken together and cholesterol but depleted of phospholipids (Brown and with the data in this study, this suggests that the SH2 and Rose, 1992; Parton and Simons, 1995). These micro- SH3 domains of LCK are not essential for intracellular domains have been termed glycolipid-enriched membranes targeting of the fully lipid-modiﬁed protein. Rather, this (GEMs) and are characterized by their insolubility in cold is achieved by dual acylation of its ﬁrst ﬁve amino acids non-ionic detergents (Rodgers et al., 1994). Some members with myristate and S-acyl moieties which directly bind of the SRC family of PTKs, including LCK, are associated LCK to the plasma membrane and perinuclear vesicles. 4992 S-acylation and signalling function of LCK LCK is associated with the co-receptors CD4 and CD8 plasma membrane. Steric hindrance by the extracellular through their cytoplasmic domains and cysteine residues domain of the 16:7:LCK-C3,5A chimera may explain why in the N-terminal unique domain of LCK, which are it was necessary to co-cross-link it with the TCR to distinct from the sites of S-acylation (Rudd, 1990). A reconstitute signalling in JCam-1.6 cells. The reconstitu- previous study demonstrated that a non-S-acylated C3S,5K tion of TCR-induced increases in intracellular free Ca mutant of LCK is not able to form a complex with CD4 in response to TCR ligation in JCam-1.6 cells was also or CD8 α when expressed in COS-7 cells, whereas singly only achieved by LCK mutants which were present at the S-acylated LCK mutants are able to complex with these plasma membrane, and the delocalized C3,5A mutant surface molecules (Turner et al., 1990). Thus, the binding was completely inactive (Figure 8A). Furthermore, the of the N-terminal unique region of LCK to CD4 or CD8 16:7:LCK-C3,5A chimera could only induce increased α correlates with the targeting of LCK to the plasma intracellular free Ca under conditions in which membrane. In addition, the COS-18 and JCam-1.6 cells phospho-ζ was formed and associated with phospho-ZAP- used in this study do not express CD4 or CD8. These 70 after co-cross-linking. Thus ζ and ZAP-70 phosphoryl- data are consistent with the hypothesis that it is the ation was closely coupled to subsequent increases in S-acylation of LCK that targets it to the plasma membrane intracellular free Ca . rather than interaction of its unique domain with cell The Sefton laboratory has also investigated the role of surface CD4 or CD8 α. lipidation in the biological activity of a constitutively One of the primary functions of LCK in TCR signalling active mutant of LCK, LCK-Y505F (Yurchak and Sefton, is to phosphorylate ITAMs in the cytoplasmic tails of the 1995). In contrast to the data from this study, a non- CD3 complex and ζ homodimers (Weiss and Littman, S-acylated C3,5S mutant of LCK-Y505F was found to be 1994; Chan and Shaw, 1996). This facilitates recruitment catalytically inactive when expressed in 208F ﬁbroblasts. of ZAP-70 PTK to the TCR, and ZAP-70 is then phos- The inactivity of this delocalized LCK-Y505F mutant phorylated by LCK and activated. Two model cell lines, correlates with its failure to be phosphorylated on Tyr394 COS-18 (Iwashima et al., 1994) and JCam-1.6 (Straus (Yurchak et al., 1996). Based on these data, this group have and Weiss, 1992), were used to study the role of LCK suggested that LCK must be associated with membranes in S-acylation in these early signalling events. Experiments order to be catalytically active, perhaps by facilitating with the heterologous COS-18 cell system indicated that Tyr394 phosphorylation. However, the present study shows phosphorylation of the CD8-ζ chimera and its association that the non-membrane-targeted C3,5A LCK mutant was with ZAP-70 occurred only with LCK mutants which highly biologically active when expressed in either COS- could localize, at least in part, to the plasma membrane 18 cells or JCam-1.6 cells. Similarly, a non-myristoylated (Figure 3). The delocalized C3,5A mutant was highly cytoplasmic form of LCK is highly active when expressed catalytically active (Figure 2B) and induced much higher in Sf9 insect cells (Carrera et al., 1991). Taken together, levels of non-speciﬁc tyrosine phosphorylation than the these data suggest that membrane attachment is only correctly localized proteins (Figure 2A), but did not required for LCK catalytic activity in certain cell types. phosphorylate CD8-ζ (Figure 3). The failure of this mutant This may reﬂect differences in the expression of either to phosphorylate the chimera was probably due to its kinases or phosphatases that act on LCK to alter its basal inaccessibility to the ITAMs of the ζ cytoplasmic tail at state of phosphorylation, thereby changing its activity. the plasma membrane. Consistent with this hypothesis, All of the LCK acylation mutants were found to be the CD16.7:LCK-C3,5A chimera, which was neither deﬁcient, to different extents, in their ability to reconstitute myristoylated nor S-acylated on LCK but which was TCR induction of two late events in JCam-1.6 cells, namely expressed efﬁciently at the plasma membrane, could CD69 and NFAT expression (Figure 5). As expected, the induce tyrosine phosphorylation of CD8-ζ when expressed C3,5A mutant, which did not reconstitute early tyrosine in COS-18 cells (Figure 6B). This suggests that one of phosphorylation stimulated by the TCR in JCam-1.6 cells, the functions of S-acylation is to target LCK to the plasma also failed to support the induction of either CD69 or membrane where it is in close proximity to the TCR. NFAT. The C3A mutant was able to support the induction Qualitatively similar results were obtained in the JCam- of CD69 and NFAT expression after TCR stimulation, but 1.6 clones in which the function of LCK acylation mutants to much lower levels than the WT protein. The C5A could be analysed in response to TCR ligation. Thus mutant was also slightly impaired in both of these late singly S-acylated LCK mutants, which were present at the responses compared with WT. The difference in function plasma membrane, were able to interact functionally with between the two singly S-acylated mutants and WT protein the TCR to induce ζ phosphorylation and its subsequent was probably due to quantitative differences in the amount association with ZAP-70 after stimulation with CD3 of LCK stably associated with the plasma membrane, antibody (Figure 4). In contrast, the delocalized C3,5A although the total amount of each mutant LCK expressed mutant completely failed to reconstitute TCR signalling. was similar to WT. This correlates well with the relative The catalytic activity of this mutant was similar to WT efﬁciency of S-acylation, which this laboratory and that LCK (Figure 2C), suggesting that its failure to complement of Lublin have found to be higher for the C5A mutant the JCam-1.6 signalling defect was due to inability to than the C3A mutant (Koegl et al., 1994; Kwong and interact with the TCR at the plasma membrane. The non- Lublin, 1995). These data, however, contrast with those acylated 16:7:LCK-C3,5A chimera was able to induce of Rodgers et al. (1994) who identiﬁed C5 as the major efﬁciently both ζ phosphorylation and its association with S-acylation site on LCK. Both of the singly S-acylated ZAP-70 after co-cross-linking of the chimera with the mutants could induce phospho-ζ and its association with TCR (Figure 7A). This again supports the hypothesis that phospho-ZAP-70 to levels comparable with WT (Figure a primary role of LCK S-acylation is to target it to the 4). The phosphorylation of the major TCR-induced 4993 P.S.Kabouridis, A.I.Magee and S.C.Ley PTyr proteins in JCam-1.6 cells transfected with the singly transfected with the chimera was reduced relative to the S-acylated mutants was also similar to WT LCK (data not WT protein (Figure 10). Since the Ras-ERK pathway is shown). The induction of CD69 and NFAT, therefore, was required for induction of CD69 and NFAT by the TCR not closely linked with the rapid tyrosine phosphorylations (D’Ambrosio et al., 1994; Genot et al., 1996), it is possible induced by TCR stimulation, and implies that LCK played that deﬁcient ERK activation by the chimera accounts an additional role in the activation process which was for its inability to support these late activation events. sensitive to its S-acylation status. However, it is possible Interestingly, anergic CD4 T cells are also deﬁcient in that the TCR-induced phosphorylation of minor PTyr their ability to activate the Ras-ERK pathway and to substrates, which were not evident when total PTyr proteins produce IL-2 following TCR stimulation (Fields et al., were analysed, was reduced with the singly S-acylated 1996; Li et al., 1996). The data in this study raise mutants, due to their inefﬁcient targeting to the plasma the possibility that an alteration of LCK function may membrane. This possibility is being investigated currently contribute to the anergic phenotype which uncouples the by two-dimensional gel electrophoresis. TCR from the efﬁcient activation of ERK MAP kinase. Yurkchak and Sefton (1995) have also investigated the Two different hypotheses may explain the partial com- importance of LCK S-acylation in T-cell function. These plementation of the JCam-1.6 signalling defect when LCK investigators tested the ability of LCK-Y505F S-acylation was artiﬁcially expressed as a transmembrane protein. mutants to induce IL-2 in stably transfected T-cell First, it was possible that LCK must detach from the hybridomas in an antigen receptor-independent fashion. plasma membrane after TCR stimulation, as a consequence These experiments failed to detect any differences between of its S-deacylation. This might be important either as a WT LCK-Y505F and singly S-acylated mutants in the mechanism to inactivate LCK or to give it access to induction of IL-2, and concluded that S-acylation of either cytosolic substrates (Milligan et al., 1995; Paige et al., Cys3 or Cys5 was sufﬁcient for full functional activity of 1995). This obviously could not occur when LCK was LCK. These data contrast with the results in this study in anchored via a transmembrane domain. However, co- which both of the singly S-acylated LCK mutants were transfection of the cytosolic C3,5A LCK mutant failed to functionally compromised relative to WT in the induction reconstitute TCR-induced NFAT production in JCam-1.6 of either NFAT or CD69 following TCR stimulation cells expressing the chimera (Figure 11B). Similarly, (Figure 5). This difference probably arises from the use an activated C3,5A LCK-Y505F mutant also did not by Sefton and colleagues of mutants which had high levels reconstitute TCR induction of NFAT in this cell line (data of constitutive activity, which were effectively uncoupled not shown). In contrast, WT LCK was able to restore from upstream regulatory events, with the result that the normal TCR signalling (Figure 11B), conﬁrming that the assay in T-hybridoma cells did not require TCR stimulation TCR signalling machinery was still intact in this cell line. to induce IL-2. In contrast, this study investigated the Taken together, these data did not support the hypothesis function of LCK acylation mutants that were not muta- that the signalling deﬁciency of the 16:7:LCK-C3,5A tionally activated in a T-cell line in which the induction chimera resulted from its inability to detach from the of IL-2 is completely dependent on TCR stimulation and plasma membrane after TCR stimulation. LCK activity. The requirements for S-acylation in LCK A second explanation to account for the signalling function, therefore, were necessarily more stringent and deﬁciency of 16:7:LCK-C3,5A was its exclusion from also more physiologically relevant. Indeed, it is not clear GEMs, in contrast to the WT protein. Thus the LCK how LCK-Y505F induces IL-2 in T hybridomas, and chimera was differentially distributed within the plane of whether this requires ζ and ZAP-70 phosphorylation. the plasma membrane relative to the WT LCK protein. Unlike the situation with the singly S-acylated LCK This may have affected the accessibility of LCK to critical mutants, the function of the LCK transmembrane chimera target proteins which were located in the GEMs. Perhaps was not limited by its level of expression at the plasma signiﬁcantly, two recent studies have suggested that Ras membrane (Figure 6C). Tyrosine phosphorylation of ZAP- is localized to caveolae, that share many properties in 70, TCR ζ and other intracellular proteins following TCR common with GEMs (Schnitzer et al., 1995; Mineo et al., stimulation was very similar between the JCam-1.6 cells 1996; Song et al., 1996). However, since lymphocytes do transfected with WT LCK or the 16:7:LCK-C3,5A chimera not contain caveolae but do contain GEMs (Fra et al., (Figure 7). The chimeric protein was also able to reconsti- 1994), the relevant plasma membrane microdomain in tute TCR-induced increases in intracellular free Ca to JCam-1.6 cells must be the latter. The exclusion of the levels greater than the WT protein (Figure 8A). Thus LCK chimera from GEMs, therefore, may prevent access retargeting of the LCK-C3,5A mutant to the plasma to critical substrates which are involved in the regulation membrane by fusing it to a transmembrane protein restored of Ras. A 36 kDa protein has been described that interacts some of its signalling functions. This is consistent with with GRB2 via its SH2 domain in T lymphocytes after the hypothesis that the primary role of S-acylation is to TCR stimulation (Buday et al., 1994; Sieh et al., 1994) target LCK to the plasma membrane where it can interact and has been postulated to couple the TCR to the activation with the TCR. However, unlike the WT protein, the of Ras. Interestingly, kinetic experiments revealed that a 16:7:LCK-C3,5A chimera was completely unable to recon- 36 kDa PTyr protein was transiently phosphorylated in stitute the induction of CD69 and NFAT by the TCR 16:7:LCK-C3,5A-expressing cells after TCR ligation, (Figure 9). The 16:7:LCK-C3,5A chimera was able to compared with a more sustained phosphorylation in cells reconstitute a sustained increase in intracellular free Ca expressing the WT protein (Figure 7C). Preliminary in the JCam-1.6 cells (Figure 8B), which is necessary for experiments have indicated that this 36 kDa PTyr protein TCR induction of IL-2 (Goldsmith and Weiss, 1988). can be precipitated with a GST–GRB2 fusion protein However, ERK MAP kinase activation in JCam-1.6 cells (data not shown). Thus it is possible that the 16:7:LCK- 4994 S-acylation and signalling function of LCK appropriate pREP3 DNA construct was then added and the cell suspension C3,5A chimera is not able to sustain prolonged phosphoryl- electroporated (Bio-Rad Gene pulser; 330 V/960 μF). The cells were ation of the GRB2-associated 36 kDa PTyr protein and, then cultured in 10 ml of complete medium for 24 h, hygromycin B as a consequence, Ras is not activated properly. This was added to a ﬁnal concentration of 0.25 mg/ml and the cells from a possibility currently is being investigated. single transfection seeded into four 96-well plates at limiting dilution (30% of wells giving rise to cell growth). Half of the medium from In conclusion, these data demonstrate an essential role each well was replaced every 3 days with fresh medium plus hygromycin for N-terminal S-acylation in the function of LCK in B, and 3 weeks later hygromycin B-resistant clones were assayed TCR signalling. This in part reﬂects a requirement for for LCK expression. Clones which expressed similar amounts of the S-acylation to target LCK to the plasma membrane where transfected LCK protein were selected for further study. it can interact with one of its critical substrates, the TCR. Antibodies However, the inability of the 16.7:LCK-C,3,5A chimera The LCK-1 anti-LCK antibody was used for confocal microscopy and to complement fully the signalling defect in the JCam-1.6 immunoprecipitations (Koegl et al., 1994). For immunoblotting, the cells indicates that the attachment of LCK to the plasma 2166 rabbit anti-LCK antibody was used, which was raised against a membrane via dual acylation of its unique domain plays denatured GST–LCK fusion protein (from Sheldon Ratnovsky, BASF, Worcester, MA). The anti-CD8 mAb, OKT8, was a kind gift from Dr some additional role which is essential for it to carry out C.Tsoukas (San Diego, CA). The anti-CD3 antibody, OKT3, was obtained its function in TCR signalling. from the American Type Culture Collection (Rockville, MD), and (Fab) fragments of this antibody were kindly prepared by A.Tutt and M.Glennie (Tenovus, Southampton, UK). Tyrosine-phosphorylated proteins were Materials and methods detected using the 4G10 anti-PTyr mAb (from Brian Druker, Oregon Health Sciences University, Portland, OR). The ZAP-4 anti-ZAP-70 Generation of LCK constructs antiserum has been described previously (Huby et al., 1995) and was A cDNA encoding wild-type mouse LCK (WT LCK) was subcloned afﬁnity puriﬁed using the immunizing peptide for immunoblotting. The neo into the KpnI–XbaI sites of the pcDNA3 expression vector (Invitrogen). TCR ζ chain was recognized in Western blots using the N39 antiserum The LCK mutants C3A, C5A and C3,5A were generated from this WT kindly provided by Jaime Sancho (Granada, Spain). The anti-CD16 mAb cDNA using PCR with the following oligonucleotides as primers: 5- and the ﬂuorescein isothiocyanate (FITC)-conjugated anti-CD69 mAb CAGGTACCATGGGCGCTGTCTGCAGCTCA-3 (C3A), 5-CAGGT- were both obtained from PharMingen. Activated ERK-1/2 was detected ACCATGGGCTGTGTCGCCAGCTCA-3 (C5A) and 5-CAGGTACC- using the PhosphoPlus p44/42 MAPK antibody from New England ATGGGCGCTGTCGCCAGCTCA-3 (C3,5A). The oligonucleotide 5- Biolabs. A rabbit antiserum which recognizes both ERKs 1 and 2 in GTCGAAGTCTCTGACCGACAG-3, containing the BamHI restriction Western blots was provided by Jeremy Tavare (University of Bristol, UK). site at position 443 of the LCK coding region, was used as the reverse primer in the PCR reaction to amplify 450 bp fragments. The various Immunoprecipitation and Western blotting analysis PCR products were digested with KpnI–BamHI restriction enzymes and Transfected COS-18 cells were washed free of serum with phosphate- then inserted into the KpnI–BamHI-treated pcDNA3/LCK construct. buffered saline (PBS) and then lysed with 1 ml of ice-cold immuno- To generate the 16:7:LCK-C3,5A chimera, the oligonucleotide 5- precipitation buffer [IPB; 150 mM NaCl, 20 mM Tris–HCl pH 7.4, 1% AAGCTTACGCGTATGGGCGCTGTCGCCAGC-3 was used as a for- NP-40, 1 mM phenylmethylsulfonyl ﬂuoride (PMSF), 100 mM Na VO , 3 4 ward primer. This oligonucleotide, which contained an MluI restriction 5 mM NaF and 5 μg/ml each of chymostatin, leupeptin and pepstatin] site 5 of the initiation ATG codon, was used for PCR in combination with scraping. JCam-1.6 cells stably expressing the various forms of the with the reverse primer described above. Following ampliﬁcation, the 7 LCK protein were resuspended to 210 cells/ml in ice-cold RPMI PCR products were digested with MluI and BamHI restriction enzymes. medium, and were left unstimulated or were stimulated for 5 min with The 16:7 fragment of the chimera was obtained from the 16:7:ZAP 1 μg/ml of OKT3 (Fab) fragments at 37°C. Cells were then pelleted chimera (Kolanus et al., 1993) following digestion with HindIII and by pulsing in a microcentrifuge and lysed in 1 ml of IPB for 15 min at MluI. The two DNA fragments were then used in a three way ligation 4°C. Cell lysates were cleared of insoluble debris by centrifugation at with a HindIII–BamHI-digested pcDNA3/LCK construct to generate the 13 000 g for 15 min at 4°C and then pre-cleared once by incubation neo 16:7:LCK-C3,5A chimera sub-cloned into the pcDNA3 expression with 10 μl of protein A–Sepharose (Pharmacia) for 15 min at 4°C. For vector. All PCR products were veriﬁed by DNA sequencing. immunoprecipitation, 10 μg of puriﬁed monoclonal antibody or 5–10 μl The various LCK mutant and chimera constructs subcloned into of antiserum were coupled covalently to 10 μl of protein A–Sepharose neo the pcDNA3 expression vector were used for transient expression with dimethylpimelimidate (Schneider et al., 1982) and incubated with experiments in COS-18 cells. To generate stable JCam-1.6 cell lines, the pre-cleared cell lysate for 4 h or overnight. Precipitation of ZAP-70 cDNA constructs were subcloned into the pREP3 episomal expression using a phospho-ITAM1 peptide coupled to Afﬁgel 10 (Bio-Rad) was vector (Hambor et al., 1988). carried out as described previously (Osman et al., 1995). Following extensive washing with ice-cold IPB, isolated protein was resolved by Cell culture and transfections SDS–PAGE and transferred onto polyvinylidene diﬂuoride (PVDF) COS-18 cell cultures were maintained in Dulbecco’s modiﬁed Eagle’s membranes. Western blotting was carried out as described previously medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM (Ley et al., 1994b). PVDF membranes were stripped of bound antibody L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The using the Amersham ECL protocol in experiments in which blots were JCam-1.6 variant of E6.1 Jurkat cells was cultured in RPMI 1640 probed for multiple antigens. medium supplemented with 5% FCS and the above concentrations of L-glutamine and antibiotics. Both cell lines were maintained in a rapid NFAT–luciferase assay growth phase prior to transfection. JCam-1.6 cells (1010 ) expressing the various LCK mutants were For transient transfections, COS-18 cells were expanded to 75% transfected by electroporation with 15 μg of pBR322-3NFAT-Luc conﬂuence in 175 cm culture ﬂasks (Nunclon) and harvested by vector (from Gerry Crabtree, Stanford, CA) using the methodology trypsinization. Detached cells were washed twice with HEPES-buffered outlined above. Cells were then cultured at 37°C for 2 h and 10 μgof saline (HBS: 140 mM NaCl, 5 mM KCl, 0.7 mM Na HPO ,20 mM OKT3 antibody added to half of the transfected cell cultures. The 2 4 HEPES pH 7.5). Approximately 510 cells, resuspended in 800 μl of remaining cultures were left unstimulated. After a further 12 h in culture, HBS, were transferred into a 0.4 cm cuvette (Bio-Rad), and the indicated cells were lysed with 150 μl of lysis buffer [0.5% NP-40, 100 mM neo amount of the appropriate pcDNA3 expression vector DNA was HK PO , 1 mM dithiothreitol (DTT) pH 7.8] for 10 min on ice. Post- 2 4 added together with sonicated salmon sperm DNA (Promega) to give a nuclear supernatants were assayed for luciferase using the Promega total of 100 μg of DNA per transfection. Cells were transfected by luciferase assay kit with a Clinilumat (Berthold) luminometer. Assays electroporation (Flowgen electroporator; 350 V, 300 μF) and then were performed in duplicate and the results shown are the mean SE. cultured for 48 h in a 10015 cm Petri dish (Nunclon) before harvesting. For the generation of stable transfectants, JCam-1.6 cells were washed Calcium analysis 6 6 twice with FCS-free RPMI 1640 medium, and 1010 cells were JCam-1.6 cells (510 ), transfected with the indicated LCK mutants, resuspended in 800 μl of the fresh serum-free medium and transferred were incubated in 1 ml of complete medium supplemented with 3 mM into an electroporation cuvette (Bio-Rad). Twenty ﬁve μg of the of the Ca indicator Indo-1 at 37°C for 1 h. Following incubation, the 4995 P.S.Kabouridis, A.I.Magee and S.C.Ley cells were washed twice with LOCKS buffer (150 mM NaCl, 1 mM microscope. Fluorescein-labelled antibody was excited at 488 nm using CaCl , 1 mM MgSO , 5 mM KCl, 10 mM glycine, 15 mM HEPES a krypton–argon mixed gas laser (Bio-Rad) and a K1 ﬁlter. Images were 2 4 pH 7.4), resuspended in the same buffer to a concentration of 110 collected using the Kalman ﬁlter settings of the COMOS programme cells/ml and then kept on ice, protected from light, until analysis. Before (Bio-Rad). Data are presented as single optical sections. analysis, the cells were pre-warmed in a 37°C waterbath for 1 min and Ca ﬂuctuations before and after the addition of the indicated ligands Cell fractionation were monitored using an LS50 Perkin Elmer Luminescence spectrometer. Transiently transfected COS-18 cells, or 1010 JCam-1.6 cells stably Cells were excited at 355 nm and emission measured at 480 and expressing LCK mutants, were resuspended in 1 ml of hypotonic buffer 405 nm, representing free versus Ca -associated Indo-1, to give an (10 mM Tris, 2 mM EDTA and 1 mg/ml each of chymostatin, leupeptin absorbance ratio. For prolonged Ca measurements, cells were loaded and pepstatin, pH 7.4), and then subjected to two successive freeze– with Indo-1 as described above with the only difference that cells were thaw cycles. The cell suspension was homogenized on ice using a washed and then cultured in a 5% FCS-containing RPMI medium. Ca Dounce homogenizer (40 strokes) and the salt concentration was then levels before and after the addition of stimulating mAbs were determined adjusted to 150 mM NaCl. Intact cells, nuclei and other debris were with a Becton Dickinson FACS Vantage. For the indicated time points, pelleted by two successive centrifugations at 480 g for 5 min. Soluble a sample of ~110 cells was analysed and the violet/blue emission and particulate fractions were generated following centrifugation at ratio was determined. When not analysed in the FACS, cell cultures 100 000 g for 30 min. Equivalent portions of the fractionated protein were were kept in a 37°C waterbath. resolved by SDS–PAGE and Western blotted with anti-LCK antibody. Flow cytometric analysis of cell surface antigens To determine cell surface expression of the 16:7:LCK-C3,5A chimera, Sucrose gradient fractionation of cell extracts immunoﬂuorescent staining was performed as described previously (Ley JCam-1.6 cells (5010 ), expressing the various LCK mutants as et al., 1994a) and cells were analysed on a Becton Dickinson FACS indicated, were washed once in PBS and then lysed for 15 min in 1 ml Vantage. To assay for the induction of CD69 expression, 110 cells of of MNE buffer (150 mM NaCl, 2 mM EDTA, 25 mM MES pH 6.5) the appropriate LCK-transfected JCam-1.6 clone were cultured in a 24- containing 1% Triton X-100 and protease inhibitors, on ice. An equal well plate (Nunclon) in the presence or absence of 10 μg/ml of the CD3 volume of an 80% sucrose solution in MNE buffer was mixed with the mAb OKT3 for 48 h. In the case of the 16:7:C3,5A chimera, stimulation lysates to form a 40% suspension, and a step sucrose gradient was was performed with CD3 plus CD16 mAbs cross-linked with the addition formed by overlaying with 2 ml of 30% sucrose–MNE and 1 ml of 5% of anti-Ig antibody. The cells were then stained with an FITC-conjugated sucrose–MNE. Isopycnic equilibriation was achieved by centrifugation CD69 mAb. Background ﬂuorescence levels were set using an FITC- at 200 000 g for 14 h in an SW55 rotor (Beckman) at 4°C. Two 2 ml conjugated mouse IgG myeloma. fractions were then collected; one from the top of the tube that contained the 30/5% sucrose interface and the other from the bottom 2 ml, which In vitro kinase assay for LCK activity contained the 40% sucrose fraction. Twenty ﬁve μl of each fraction was COS-18 cells were washed twice with PBS and then lysed in 1 ml of mixed with an equal volume of 2 Laemmli sample buffer and the of ice cold buffer (SB) comprising 50 mM N-octyl-β-D-glucopyranoside proteins were separated by 12% SDS–PAGE, transferred onto PVDF (Sigma), 150 mM NaCl, 25 mM Tris, 1 mM Na VO , 2 mM Na P O , membrane and then probed with anti-LCK antibody. 3 4 4 2 7 20 mM NaF and 1 μg/ml each of chymostatin, leupeptin and pepstatin, pH 7.2. Cell lysates were cleared of insoluble material by centrifugation at 13 000 g for 10 min. Precipitation with anti-LCK antibody coupled Acknowledgements to protein A–Sepharose for 4 h was performed as described above. Immunoprecipitates were then washed ﬁve times in lysis buffer and The authors would like to thank A.Weiss, G.Crabtree, M.Tykocinski, once in kinase buffer (100 mM NaCl, 50 mM HEPES pH 7.5, 5 mM S.Ratnovsky and D.Littman for the reagents used in this study. The MgCl , 5 mM MnCl and1mM Na VO ). Each immunoprecipitate was 2 2 3 4 authors are also grateful to the Photo-Graphics department at NIMR and then resuspended in 20 μl of kinase buffer supplemented with 10 μM to E.Hirst for their help with the ﬁgures. This work was supported by ATP, 5 μCi [ P]ATP and 1 mM RR-SRC peptide substrate (Gibco- the Medical Research Council. BRL) and incubated at room temperature for 30 min. The reaction was stopped by addition of 30 μl of kinase buffer plus 25 mM EDTA and then peptide phosphorylation was measured by binding to p81 paper (Whatman) and Cerenkov counting. References To assay the activity of LCK expressed in the JCam-1.6 clones, cells Appleby,M.W., Gross,J.A., Cooke,M.P., Levin,S.D., Qian,X. and were lysed using SB containing 1% Triton X-100 instead of N-octyl-β- Perlmutter,R.M. (1992) Defective T cell receptor signaling in mice D-glucopyranoside. Immunoprecipitation was carried out as for COS-18 fyn lacking the thymic isoform of p59 . 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Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1997
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/16.16.4983
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.16 No.16 pp.4983–4998, 1997 S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes 1,2 tion by interacting sequentially with two different families Panagiotis S.Kabouridis , 2,3 1,3 of cytoplasmic non-receptor PTKs. Anthony I.Magee and Steven C.Ley The ﬁrst of these is the SRC family, in particular LCK 1 2 Divisions of Cellular Immunology and Membrane Biology, and FYN (Perlmutter et al., 1993). Analysis of LCK- National Institute for Medical Research, The Ridgeway, Mill Hill, deﬁcient T-cell lines has indicated that LCK is absolutely London NW7 1AA, UK required for TCR signalling (Karnitz et al., 1992; Straus Corresponding authors and Weiss, 1992). Indeed, in the absence of LCK, the TCR fails to induce any tyrosine phosphorylation of LCK is a non-receptor protein tyrosine kinase required cytoplasmic proteins, and all downstream signalling events for signal transduction via the T-cell antigen receptor are blocked. Consistent with its important role in TCR (TCR). LCK N-terminus is S-acylated on Cys3 and signalling, T cell development is severely impaired in Cys5, in addition to its myristoylation on Gly2. Here LCK null mice (Molina et al., 1992). Genetic studies have the role of S-acylation in LCK function was examined. also indicated that FYN is involved in TCR signalling in Transient transfection of COS-18 cells, which express mature thymocytes (Appleby et al., 1992; Stein et al., a CD8-ζ chimera on their surface, revealed that LCK 1992) and, in certain T-cell lines, may be associated with mutants that were singly S-acylated were able to target the TCR (Samelson et al., 1990). However, from the to the plasma membrane and to phosphorylate CD8-ζ. phenotype of FYN null mice, it appears that the role of A non-S-acylated LCK mutant did not target to the FYN in TCR signalling may be restricted to particular plasma membrane and failed to phosphorylate CD8-ζ, stages of T-cell development (Appleby et al., 1992; Stein although it was catalytically active. Fusion of non-S- et al., 1992). acylated LCK to a transmembrane protein, CD16:7, The second family of PTKs which interact with the allowed its plasma membrane targeting and also TCR comprises ZAP-70 (Chan et al., 1992) and SYK phosphorylation of CD8-ζ when expressed in COS-18 (Taniguchi et al., 1991). After stimulation, ZAP-70 and cells. Thus S-acylation targets LCK to the plasma SYK are recruited to phosphorylated CD3 and ζ subunits membrane where it can interact with the TCR. When of the TCR, where they are in turn tyrosine phosphorylated expressed in LCK-negative JCam-1.6 T cells, delocal- (Chan et al., 1992, 1994b; Straus and Weiss, 1993; Wange ized, non-S-acylated LCK was completely non-func- et al., 1993; Chan and Shaw, 1996). ZAP-70 and SYK tional. Singly S-acylated LCK mutants, which were bind to the TCR via their two N-terminal SH2 domains expressed in part at the plasma membrane, efﬁciently which interact with speciﬁc phosphorylated tyrosines of reconstituted the induced association of phospho-ζ with the cytoplasmic tails of the CD3 complex (γδε) and ζ ZAP-70 and intracellular Ca ﬂuxes triggered by the dimers in immunoreceptor tyrosine-based activation motifs TCR. Induction of the late signalling proteins, CD69 (ITAMs) (Chan et al., 1994a; Chan and Shaw, 1996). and NFAT, was also reconstituted, although at reduced In an LCK-deﬁcient T cell line, TCR ITAMs are not levels. The transmembrane LCK chimera also sup- phosphorylated and ZAP-70 is not recruited to the TCR ported the induction of tyrosine phosphorylation and after stimulation, indicating that ZAP-70 plays a role Ca ﬂux by the TCR in JCam-1.6 cells. However, downstream of LCK (Straus and Weiss, 1992; Iwashima induction of ERK MAP kinase was reduced and the et al., 1994). Studies in COS cells have suggested that chimera was incapable of reconstituting induced CD69 one function of LCK is to phosphorylate ITAMs of the or NFAT expression. These data indicate that LCK TCR, thereby mediating association of the receptor with must be attached to the plasma membrane via dual ZAP-70 and its subsequent phosphorylation by LCK acylation of its N-terminus to function properly in (Iwashima et al., 1994; Chan et al., 1995) Consistent with TCR signalling. this hypothesis, both constitutive and inducible tyrosine Keywords: LCK/localization/S-acylation/signalling phosphorylation of ζ is abolished in LCK null thymocytes (van Oers et al., 1996). Tyrosine phosphorylation of ZAP- 70 activates its kinase activity (Chan et al., 1995; Wange et al., 1995) and, together with LCK, mediates tyrosine Introduction phosphorylation of multiple intracellular proteins which trigger T-cell proliferation. An essential role for ZAP-70 The induction of protein tyrosine kinase (PTK) activity in TCR signalling and T-cell development has been by the T-cell antigen receptor (TCR) is essential to couple it to downstream pathways which trigger proliferation and revealed by genetic studies in both murine and human differentiation of resting T cells into effector T cells systems (Hivroz and Fischer, 1994; Negishi et al., 1995). (Weiss and Littman, 1994). However, the component In contrast, SYK does not appear to be essential for αβ subunits of the TCR do not contain any intrinsic tyrosine T cell development (Turner et al., 1995), although the kinase domains. Rather, the TCR initiates signal transduc- development of epithelial γδ T cells is disrupted in SYK- © Oxford University Press 4983 P.S.Kabouridis, A.I.Magee and S.C.Ley Table I. Schematic representation of LCK acylation mutants Myr Pal Pal ↓↓ ↓ WT Met Gly Cys Val Cys Ser Ser Asn Pro Glu Asp C3A Met Gly Ala Val Cys Ser Ser Asn Pro Glu Asp C5A Met Gly Cys Val Ala Ser Ser Asn Pro Glu Asp C3,5A Met Gly Ala Val Ala Ser Ser Asn Pro Glu Asp The ﬁrst 11 amino acids of murine LCK are shown in three letter code, with amino acids that are modiﬁed by myristoylation (Myr) or S-acylation (Pal) in bold type. Mutated amino acids are shown in italics. negative mice (Mallick-Wood et al., 1996). The precise (C5A) or both cysteines (C3,5A) were substituted by an role of SYK in signalling via the TCR is presently unclear. alanine (Table I). The C3A, C5A and C3,5A mutants are However, recent experiments have indicated that SYK has myristoylated but they lack one (C3A and C5A) or both different activation requirements to ZAP-70 and does not (C3,5A) of the S-acyl attachment sites. require LCK expression (Chu et al., 1996). Thus it is Localization of the LCK mutants was ﬁrst analysed by likely that ZAP-70 and SYK play distinct roles in TCR transient expression in COS-18 cells and subcellular signalling. fractionation. Wild-type (WT) LCK was found exclusively Many proteins are now known to be post-translationally in the particulate fraction (Figure 1A). The majority of modiﬁed by the covalent addition of lipid moieties (Casey, the singly S-acylated C3A and C5A mutants, 71 and 92% 1995; Milligan et al., 1995). Prominent among these are respectively, was also found in the particulate fraction. In proteins involved in signalling via cell surface receptors, contrast, the minority of the C3,5A mutant was detected including the α and γ subunits of heterotrimeric G proteins in the particulate fraction (33%). This crude analysis (Milligan et al., 1995), small GTP-binding proteins such suggested that S-acylation of LCK was affecting its as Ras (Newman and Magee, 1993) and the SRC family localization. This was analysed in more detail by of protein tyrosine kinases (Resh, 1994). These lipid immunoﬂuorescence and confocal microscopy. In these modiﬁcations have been found to play key roles in experiments, transfected COS-18 cells were ﬁrst made association of these otherwise hydrophilic proteins with non-adherent and spherical by vigorous pipetting to facilit- the cytoplasmic face of speciﬁc cellular membranes. The ate detection of plasma membrane staining. WT LCK was N-terminal unique domain of LCK is modiﬁed by the detected exclusively at the plasma membrane, whereas addition of two different types of lipid (Shenoy-Scaria the C3,5A mutant was found diffusely throughout the et al., 1993; Koegl et al., 1994; Rodgers et al., 1994). cytoplasm and also in the nucleus (Figure 1C). The C3A Myristate, a saturated acyl group of 14 carbons, is added and C5A mutants showed an intermediate distribution and co-translationally to Gly2 via an amide bond, replacing were detected both at the plasma membrane and in the the initiator methionine (Johnson et al., 1994). Post- cytoplasm. translationally, two longer chain fatty acyl groups, which The role of acylation in LCK localization in T cells are often C16 palmitates, are attached by labile thioester was also investigated by stably expressing the panel of bonds to Cys3 and Cys5 (Milligan et al., 1995). In this mutants in a derivative of the Jurkat T-cell line, JCam- study, the role of S-acylation of LCK has been investigated 1.6, which expresses low levels of a catalytically inactive by analysis of point mutants expressed in COS-18 cells deletion mutant of LCK (Straus and Weiss, 1992). Sub- and in a leukaemic T-cell line which does not express cellular fractionation revealed that WT LCK was almost functional LCK. These experiments indicate that LCK exclusively in the particulate fraction (93%), similar to S-acylation is required for it to couple the TCR to COS-18 cells (Figure 1B). Removal of one or both downstream signalling pathways which, in part, reﬂects its S-acylation sites shifted the LCK into the soluble fraction. role in correct targeting of LCK to the plasma membrane. This shift was particularly pronounced with the C3A mutant where the majority of the protein (69%) was detected in the soluble fraction. Immunoﬂuorescence and Results confocal microscopy demonstrated that the WT protein N-terminal S-acylation is required for cortical was localized to the plasma membrane and to perinuclear targeting of LCK vesicles (Figure 1D), as shown previously (Ley et al., One of our laboratories has demonstrated previously that 1994a). The C3A and C5A mutants were also localized LCK is localized predominantly to the plasma membrane to the plasma membrane, although a signiﬁcant fraction and also to peri-centrosomal vesicles in human T lympho- was also detected diffusely distributed throughout the cytes (Ley et al., 1994a). The unique region of LCK is cytoplasm. In contrast, the C3,5A mutant was not detected modiﬁed by the addition of a myristate group to Gly2 at the plasma membrane but was present throughout the which is added co-translationally (Johnson et al., 1994). cytoplasm. Taken together, these data demonstrated that Recent studies from this and other laboratories have S-acylation was important in targeting LCK to the plasma indicated that the LCK unique region is also modiﬁed by membrane in both COS-18 and J-Cam-1.6 cells. the attachment of S-acyl groups to Cys3 and Cys5 (Shenoy- Scaria et al., 1993; Koegl et al., 1994; Rodgers et al., N-terminal S-acylation is not required for the 1994). To investigate the role of S-acylation of the LCK enzymatic activity of LCK unique region in its localization and function, three point Before analysing the signalling function of the LCK mutants were generated in which either Cys3 (C3A), Cys5 mutants, it was important to determine whether S-acylation 4984 S-acylation and signalling function of LCK Fig. 1. Subcellular localization of LCK mutants expressed in COS-18 and JCam-1.6 T cells. COS-18 cells (A and C) were transiently transfected with the LCK cDNA constructs indicated. JCam-1.6 cells (B and D) were stably transfected with plasmids encoding each of the panel of LCK mutants. Clones were then isolated in which the level of transfected LCK was similar or greater than transfected WT LCK. (A and B) Cells were disrupted by freeze–thawing and homogenization and then separated into soluble (S) and particulate (P) fractions by ultracentrifugation. Equal aliquots from the two fractions were then resolved by 10% SDS–PAGE, transferred onto PVDF membrane and probed with anti-LCK antibodies. The band that represents the transfected LCK is shown in brackets. In (B) the lower band represents the truncated, non-functional LCK form present in JCam-1.6 cells. (C and D) Cells were ﬁxed with paraformaldehyde, permeabilized and then stained for expression of LCK. The images shown are single confocal sections through representative transfected cells. Untransfected cells gave no detectable staining (data not shown). was required for LCK enzymatic activity. To investigate the mutants isolated from transfected J-Cam-1.6 T cells this, expression vectors encoding each mutant were trans- were found to have a speciﬁc activity similar to WT LCK fected into COS-18 cells which were then cultured for protein, as was found with transfected COS-18 cells 48 h. Total cell lysates were then resolved by SDS–PAGE (Figure 2C). and probed for phosphotyrosyl (PTyr) proteins. Figure 2A indicates that expression of each of the LCK mutants A non-S-acylated LCK mutant cannot resulted in induction of several PTyr proteins compared phosphorylate a CD8-ζ chimera expressed in with empty vector control. The most prominent of these COS-18 cells was a polypeptide of ~55 kDa which probably corres- COS-18 cells stably express on their surface a chimera ponded to transfected LCK itself. The C3,5A mutant, comprising the extracellular and transmembrane portions which was localized predominantly in the cytosol, was of the human CD8α chain fused to the cytoplasmic domain particularly active in the induction of PTyr proteins, many of the TCR ζ chain (Iwashima et al., 1994). Transient of which had mobilities distinct from those induced by expression of WT LCK in COS-18 cells results in phos- the plasma membrane-targeted LCK mutants. These data phorylation of the CD8-ζ chimera on tyrosines within indicated that there was no requirement for S-acylation ITAMs of the cytoplasmic tail of ζ. Co-expression of for LCK enzymatic activity in vivo. In addition, these ZAP-70 with LCK results in association of ZAP-70 with results suggested that the delocalized C3,5A LCK mutant phosphorylated ITAMs and its tyrosine phosphorylation. was accessible to distinct substrates from both the WT COS-18 cells, therefore, provide a simpliﬁed heterologous protein and singly S-acylated mutants that were associated model to study sequential interactions between the TCR with the plasma membrane. To assay the activity of LCK and the two non-receptor PTKs, LCK and ZAP-70. directly, the various mutants were immunoprecipitated As a ﬁrst step to investigate the role of LCK S-acylation from transfected COS-18 cells and tested for their ability in its functional interaction with the TCR, COS-18 cells to phosphorylate RR-SRC peptide in vitro. The RR-SRC were transfected with cDNAs encoding the various LCK peptide corresponds to residues 111–122 of SRC and mutants with or without ZAP-70 cDNA. As shown in contains its auto-phosphorylation site (Wong and Figure 3A (left side), WT LCK and singly S-acylated Goldberg, 1983). As shown in Figure 2B, all the C3A and C5A mutants were able to phosphorylate the S-acylation mutants had enzymatic activity comparable CD8-ζ chimera. In contrast, the C3,5A mutant was unable with the WT molecule. to increase the tyrosine phosphorylation of the chimera The LCK mutants were also immunoprecipitated from above background levels although this mutant was the panel of stably transfected JCam-1.6 cell lines and expressed at levels comparable with WT LCK (Figure assayed for their activity in vitro. To increase sensitivity 3B). ZAP-70 associated with the CD8-ζ chimera, resulting in these assays, a different peptide, referred to as C-peptide, in its increased tyrosine phosphorylation, when co- was used, which has been demonstrated to be the optimal expressed with WT LCK or either of the two singly substrate for LCK (Songyang and Cantley, 1995). All of S-acylated mutants (Figure 3A, right side). The non- 4985 P.S.Kabouridis, A.I.Magee and S.C.Ley Fig. 3. Induction of CD8-ζ phosphorylation by LCK mutants in COS-18 cells. COS-18 cells were transiently transfected with vectors encoding the various LCK mutants in the presence and absence of ZAP-70 cDNA. (A) CD8-ζ was immunoprecipitated from cell lysates using the OKT8 mAb, resolved by 10% SDS–PAGE and transferred electrophoretically to PVDF membrane. The blot was then probed sequentially with an anti-PTyr mAb (PY) followed by an anti-ζ antibody (zeta). The positions of phospho-ZAP-70 (ZAP-70-PO ) and phospho-CD8-ζ (CD8-ζ-PO ) are indicated. H denotes the heavy chain of the immunoprecipitating antibody. (B) Equivalent amounts of total cell lysates from the transfected COS-18 cells used in (A) were resolved by SDS–PAGE. The expression of transfected LCK and ZAP-70 was then checked by sequential immunoblotting of the PVDF membrane with the appropriate antibodies. S-acylated C3,5A mutant, however, was again unable to induce phosphorylation of CD8-ζ or its association with ZAP-70 in the doubly transfected cells. Western blot analysis demonstrated that this mutant and the co-trans- fected ZAP-70 were expressed at levels similar to that achieved in the WT LCK transfection (Figure 3B). In conclusion, these experiments indicated that non-S- acylated LCK, which did not stably interact with the Fig. 2. LCK S-acylation mutants are enzymatically active both in vivo plasma membrane, also failed to phosphorylate CD8-ζ and in vitro.(A) Lysates were prepared from COS-18 cells transiently when expressed in COS-18 cells. transfected with plasmids encoding the various LCK acylation mutants, as indicated. Proteins were resolved by SDS–PAGE, transferred onto PVDF membrane and immunoblotted with anti-PTyr Non-S-acylated LCK cannot reconstitute early mAb. The PVDF membrane was then stripped and reprobed with signalling events triggered by the TCR in JCam-1.6 anti-LCK antiserum to conﬁrm expression of the transfected LCK T cells protein (not shown). (B) LCK mutants from transiently transfected JCam-1.6 is a mutant derivative of the Jurkat T-cell line COS-18 cells were immunoprecipitated and assayed for their ability to phosphorylate RR-SRC peptide in vitro. Results shown are normalized which expresses low levels of a mutant form of LCK against the amount of LCK in immunoprecpitates as determined by that is catalytically inactive (Straus and Weiss, 1992). immunoblotting, and are expressed in arbitrary units. Data are the Stimulation of JCam-1.6 T cells with CD3 antibody fails mean ( SE) of duplicate assays. (C) LCK was immunoprecipitated to induce early or late signalling events triggered by the from JCam-1.6 clones expressing the indicated LCK mutants and assayed for its ability to phosphorylate C-peptide. Results are TCR. TCR signalling, however, may be reconstituted by presented as in (B). transfection of JCam-1.6 cells with WT LCK. To investi- 4986 S-acylation and signalling function of LCK Fig. 4. Analysis of early signalling events triggered by the TCR in LCK-transfected JCam-1.6 cells. (A) Each of the clones was stimulated for 5 min with 1 μg/ml of F(ab) fragments of the CD3 antibody OKT3 or left unstimulated and then lysed in 1% NP-40 lysis buffer. ZAP-70 was then isolated from cell lysates by immunoprecipitation and resolved by 12.5% SDS–PAGE. Tyrosine-phosphorylated proteins were detected by immunoblotting with an anti-PTyr mAb. The positions of phospho-ZAP-70 and phospho-ζ are indicated on the left of the panel. To check that equivalent levels of ZAP-70 were present in each immunoprecipitate, the PVDF membrane was stripped and probed with an anti-ZAP-70 antibody. Qualitatively similar results were obtained with at least two other clones for each of the LCK mutants (data not shown). (B) Transfected JCam-1.6 cells were loaded with the Ca -interacting agent Indo-1 and then stimulated with OKT3 F(ab) antibody. Changes in the the level of intracellular free Ca are shown as a function of time. Successful loading with Indo-1 was conﬁrmed by subsequently treating the cells with ionomycin. The times at which CD3 antibody and ionomycin were added are indicated. gate the role of LCK acylation in signalling via the TCR although several bands were constitutively phosphorylated in T cells, JCam-1.6 cells were transfected with various when compared with the empty vector control (data not LCK mutants and stable clones expressing levels of shown). Thus, S-acylation of LCK was essential for the transfected LCK similar to or greater than that of WT TCR to induce tyrosine phosphorylation of any intra- LCK were selected for analysis. For each LCK mutant, cellular proteins. qualitatively similar results to those shown below were The panel of JCam-1.6 clones was also analysed for obtained with other clones tested (data not shown). their ability to induce increases in intracellular free Ca As discussed in the Introduction, TCR cross-linking following TCR cross-linking. Both singly S-acylated LCK rapidly induces LCK to phosphorylate ITAMs of the ζ mutants induced Ca levels similar to that achieved in chain (Weiss and Littman, 1994). ZAP-70 is then recruited the WT clone (Figure 4B). However, non-S-acylated to the TCR via binding of its two N-terminal SH2 domains C3,5A mutant failed to induce any increase in intracellular and is itself tyrosine phosphorylated. To investigate the free Ca following TCR cross-linking. Taken together, importance of LCK S-acylation in these early signalling these data indicated that the LCK S-acylation mutants events, the various JCam-1.6 clones were stimulated which were at least in part targeted to the plasma membrane with F(ab) fragments of the mitogenic CD3 monoclonal were able to reconstitute early signalling events triggered antibody OKT3 for 5 min and ZAP-70 protein was by the TCR. In contrast, the non-S-acylated C3,5A mutant, immunoprecipitated from cell lysates. Introduction of WT which was completely delocalized, was non-functional. LCK or either of the singly S-acylated mutants into JCam- 1.6 reconstituted the ability of the TCR to induce tyrosine LCK S-acylation mutants are defective in phosphorylation of ZAP-70 and its association with the reconstituting late signalling events triggered by TCR (Figure 4A). In contrast, non-S-acylated C3,5A LCK the TCR in JCam-1.6 cells failed to reconstitute inducible phosphorylation of ZAP- Late activation events following stimulation of the TCR 70 or its association with phospho-ζ, although this clone include induction of the cell surface activation antigen, expressed high levels of transfected LCK. CD69 (Testi et al., 1989), and of the T cell-speciﬁc Similarly to the results with ZAP-70 tyrosine phos- transcription factor, NFAT, which is involved in transcrip- phorylation, TCR cross-linking of cell lines expressing tional regulation of the interleukin-2 (IL-2) gene (Jain singly acylated LCK induced qualitatively similar patterns et al., 1995). In order to investigate the ability of the of total PTyr proteins to the WT LCK transfectant (data various LCK mutants to support the ﬁrst of these late not shown). In contrast, the C3,5A LCK mutant failed activation events in JCam-1.6 cells, the panel of clones to support any TCR-induced tyrosine phosphorylation, was stimulated with CD3 antibody for 48 h, following 4987 P.S.Kabouridis, A.I.Magee and S.C.Ley mutant was slightly impaired relative to WT in the induction of NFAT, whereas the C3A mutant induced only 30% of WT levels of NFAT following CD3 stimulation. The C3,5A mutant was essentially inactive. All transfected cell lines induced similar levels of NFAT expression in response to stimulation with Ca ionophore and phorbol ester (data not shown). In conclusion, the experiments in this section indicate that both singly S-acylated LCK mutants were able to reconstitute late signalling pathways triggered by the TCR. However, neither of these mutants was as effective as the WT LCK protein at inducing these late signalling events. This suggested that all three attached lipid groups were required for LCK to work with maximum efﬁciency in reconstituting TCR signalling in the JCam-1.6 cells. The non-S-acylated C3,5A mutant was not capable of coupling the TCR to late signalling events, as expected from its inability to induce early signalling events following TCR cross-linking. Retargeting of non-S-acylated LCK to the plasma membrane reconstitutes early TCR-induced signalling events in JCam-1.6 cells The experiments in both COS-18 cells and JCam-1.6 cells revealed a correlation between plasma membrane targeting of LCK mutants and their ability to interact functionally with the TCR. These results, therefore, suggested that one of the primary functions of LCK S-acylation is to target it to the plasma membrane. To investigate this hypothesis, Fig. 5. Analysis of late signalling events triggered by the TCR in LCK-transfected JCam-1.6 cells. (A) A total of 110 cells from each the function was tested of LCK which could be expressed of the JCam-1.6 clones were cultured in the presence of 10 μg/ml at the plasma membrane independently of its lipid modi- OKT3 F(ab) antibody for 48 h. CD69 expression was then ﬁcations. A chimera was constructed by fusing the entire determined by immunoﬂuorescence and ﬂow cytometry. The LCK coding sequence to the extracellular domain of percentage of cells which were CD69 positive was determined using an FITC-conjugated mouse myeloma to set the background levels of CD16 and the transmembrane domain of CD7 to generate staining. Similar results were obtained in two other experiments. 16:7:LCK-WT, as shown in Figure 6A (Kolanus et al., (B) LCK-expressing JCam-1.6 cells were transiently transfected with 1993; kindly provided by Brian Seed, Boston, MA). This 15 μg of the NFAT–luciferase reporter construct, pBR322-3NFAT- protein contains intact S-acylation sites on LCK but cannot Luc. The cells were then cultured for 2 h and stimulated with be myristoylated. A mutant of this chimera was generated 10 μg/ml OKT3 antibody () or left unstimulated (–). After a further 12 h in culture, cells were lysed and luciferase activity assayed. Data by PCR in which the two S-acylation sites on LCK were are presented in arbitrary units as a mean of duplicate measurements mutated to generate the 16:7:LCK-C3,5A chimera. In vivo ( SE). Qualitatively similar results were obtained in three separate labelling with [ H]palmitate conﬁrmed that the 16:7:LCK- experiments. C3,5A chimera was not S-acylated, in contrast to 16:7:LCK-WT (data not shown). The functional experi- which the cells were analysed for CD69 expression by ments described below were all carried out on the non- immunoﬂuorescence and ﬂow cytometry. WT LCK acylated 16:7:LCK-C3,5A chimera. Similar results were induced expression of CD69 as expected (Figure 5A). obtained with 16:7:LCK-WT chimera (data not shown). Both singly S-acylated mutants were able to induce the In initial experiments, cDNAs encoding WT LCK, LCK expression of CD69, although the level of induced expres- C3,5A and the 16:7:LCK-C3,5A chimera were transiently sion in C3A mutant-transfected cells was markedly expressed in COS-18 cells and tested for their ability to reduced compared with WT LCK. The C3,5A mutant was phosphorylate CD8-ζ. As shown previously, WT LCK, completely unable to support the induction of CD69 but not the C3,5A mutant, could phosphorylate CD8-ζ. expression following TCR stimulation. However, the 16:7:LCK-C3,5A chimera was able to induce In order to assay the induction of NFAT transcription high levels of tyrosine phosphorylation of CD8-ζ (Figure factor, each JCam-1.6 clone was transfected with a con- 6B). Thus retargeting to the plasma membrane overcame struct containing the luciferase reporter gene under the the acylation requirement for LCK to interact functionally control of three copies of the NFAT regulatory element with CD8-ζ when expressed in COS-18 cells. Co-transfec- (Verweij et al., 1990). After 2 h in culture, cells were tion of ZAP-70 cDNA with 16:7:LCK-C3,5A cDNA also stimulated with CD3 antibody, or left unstimulated, and resulted in the association of ZAP-70 with CD8-ζ and its recultured for a further 12 h before harvesting. Cell lysates tyrosine phosphorylation (data not shown). were then prepared and luciferase activity assayed. A To study the function of 16:7:LCK-C3,5A in T cells, similar pattern of responsiveness was seen as with CD69 this cDNA construct was stably transfected into JCam- expression. Thus WT LCK induced NFAT following CD3 1.6 cells and an oligoclonal population of cells isolated, cross-linking as expected. The singly S-acylated C5A using ﬂuorescence activated cell sorting (FACS), which 4988 S-acylation and signalling function of LCK Fig. 6. Analysis of 16:7:LCK-C3,5A LCK chimera expressed in COS-18 and JCam-1.6 cells. (A) Schematic depiction of the 16:7:LCK-C3,5A chimera used in this study. (B) COS-18 cells were transfected with the indicated expression vectors and then CD8-ζ immunoprecipitated from cell lysates and immunoblotted sequentially for PTyr and ζ. The position of phospho-CD8-ζ is indicated on the left of the panel. Expression of transfected LCK or 16.7:LCK chimera was conﬁrmed by immunoblotting of aliquots of total cell lysate used for immunoprecipitation, with an anti-LCK antibody (data not shown). (C) The expression level of the 16:7:C3,5A LCK chimera in stably transfected JCam-1.6 cells was determined by ﬂow cytometry (dashed line). The background ﬂuorescence was set with an FITC-conjugated anti-Ig antibody (solid line). Fig. 7. Analysis of early tyrosine phosphorylation events triggered by the 16:7:LCK-C3,5A chimera in transfected JCam-1.6 cells. (A) JCam-1.6 cells stably expressing on their surface the 16:7:LCK-C3,5A chimera were stimulated with CD3 and/or CD16 antibodies in the presence of anti-IgG antibody for 5 min or left unstimulated. Control JCam-1.6 cells transfected with WT or C3,5A LCK were stimulated with CD3 plus anti-Ig antibodies. ZAP-70 was then immunoprecipitated from cell lysates and immune complexes were resolved by 12.5% SDS–PAGE and immunoblotted with anti-PTyr mAb (upper panel). The positions of phospho-ZAP-70 (ZAP-70-PO ) and phospho-ζ (ζ-PO ) are indicated on the left of the panels. 4 4 Blots were re-probed with anti-ZAP-70 antibody to conﬁrm that equivalent amounts of antigen were immunoprecipitated from each of the JCam-1.6 clones (lower panel). (B) A total of 1010 cells of the WT- or 16:7:LCK-C3,5A-expressing JCam-1.6 cells were left untreated or were stimulated as in (A). Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with the anti-PTyr mAb 4G10, resolved by SDS–PAGE electrophoresis and detected with Western blotting using anti-PTyr mAb. The asterisk indicates the migration distance of the 16:7: LCK-C3,5A chimera. (C) LCK WT- and 16:7:LCK-C3,5A-expressing JCam-1.6 cells were stimulated as in (A) for the indicated times, and cell lysates representing 0.510 cells were analysed by 10% SDS–PAGE and probed with anti-phosphotyrosine antibodies. The asterisk indicates the 16:7:LCK-C3,5A chimera while the arrow shows the activation-induced 36 kDa phosphoprotein. 4989 P.S.Kabouridis, A.I.Magee and S.C.Ley Fig. 9. Analysis of late signalling events triggered by the Fig. 8. Short and long term Ca ﬂuxes in WT- and 16:7:LCK-C3,5A- 16:7:LCK-C3,5A chimera in transfected JCam-1.6 cells. (A) A total of expressing cells. (A) The indicated transfected JCam-1.6 cells were 110 cells of each of the indicated JCam-1.6 clones were cultured loaded with Indo-1 and then sequentially stimulated with CD16 with CD3 antibody, CD16 antibody, or both, as indicated, plus anti- followed by CD3 and anti-IgG (goat anti-mouse) antibodies. IgG antibody for 48 h, or left unstimulated. The percentage of cells Fluctuations in the levels of intracellular free Ca before and after which expressed CD69 on their surface was then determined as in antibody stimulation are shown as a function of time. Successful Figure 5. (B) Transfected JCam-1.6 cells were transiently loading with Indo-1 was conﬁrmed by subsequently treating the cells transfected with 15 μg of the NFAT–luciferase reporter construct, with ionomycin (not shown). The times at which antibodies were pBR322-3NFAT-Luc. The cells were then cultured for2hand added are indicated. (B) The WT- and 16:7:C3,5A-expressing clones stimulated with CD3 antibody, CD16 antibody, or both in the presence were loaded with Indo-1 as in (A) and basal and stimulation-induced of anti-IgG antibody. After a further 12 h in culture, cells were lysed intracellular Ca levels were monitored in a FACS. For each time and luciferase activity assayed. Data are presented in arbitrary units as point, ~110 cells were analysed and the violet/blue emission ratio a mean of duplicate measurements ( SE). was determined. All the values collected were plotted as the emission ratio versus time. This is one of two experiments performed with similar results. 1.6 cells cells expressing WT LCK were stimulated with showed high levels of surface CD16 staining (Figure 6C). CD3 mAb and anti-Ig (Figure 8A). A kinetic experiment Stimulation of the TCR on cells expressing 16:7:LCK- also demonstrated that the LCK chimera was able to C3,5A failed to induce tyrosine phosphorylation of ZAP- sustain TCR-induced intracellular Ca levels above basal 70 or its association with the ζ chain (Figure 7A). In levels for upto 2 h post-stimulation, similarly to WT LCK contrast, WT LCK induced both of these events as (Figure 8B). These results, therefore, suggested that there expected. However, if the chimera was stimulated with was no requirement for S-acylation of the plasma mem- both CD3 and CD16 antibodies co-cross-linked with anti- brane retargeted LCK chimera to induce early signalling Ig, ZAP-70 phosphorylation and association with the ζ events triggered by the TCR. However, analysis of CD69 chain was strongly induced. Control experiments demon- and NFAT induction revealed that the chimera was unable strated that addition of CD3 and CD16 antibodies in to induce these late activation events, even when the TCR the absence of anti-Ig was insufﬁcient to reconstitute and CD16 were co-aggregated on the cell surface (Figure signalling, suggesting that the chimera had to be brought 9A and B). In contrast, the WT LCK-expressing clone into close proximity with the TCR in order to function was able to induce both of these events, as expected. (data not shown). The chimera was also able to reconstitute Thus the chimeric transmembrane form of LCK did not rapid increases in intracellular free Ca when the TCR reconstitute TCR coupling to late signalling events in was co-cross-linked with CD16. This increase consistently was found to be greater than that achieved when JCam- JCam-1.6 T cells. 4990 S-acylation and signalling function of LCK Fig. 10. Kinetics of ERK activation following stimulation of WT- and 16:7:LCK-C3,5A-expressing JCam-1.6 cells. WT- and 16:7:LCK-C3,5A- expressing JCam-1.6 cells were left untreated or were stimulated with OKT3 or OKT3 plus CD16 mAbs respectively, followed by cross-linking with anti-Ig antibody for the indicated times, and the phosphorylation of ERK-1 and ERK-2, in equal cell lysate aliquots, was detected by immunoblotting with anti-phospho-ERK antibody (upper panel). The same membrane was stripped and reprobed with anti-ERK-1/2 antiserum (lower panel). The migrating distances for ERK-1 and ERK-2 are indicated with arrows. The slower migrating species observed in the lower panel are indicative of ERK activation. The 16:7:LCK-C3,5A chimera is deﬁcient in its ability to reconstitute TCR-induced ERK activation in JCam-1.6 cells The failure of the 16:7:LCK-C3,5A chimera to reconstitute late signalling events triggered by the TCR was not due to a quantitative reduction in the level of phosphorylation of ZAP-70 or its association with phospho-ζ (Figure 7A). To investigate whether the chimera was able to reconstitute the tyrosine phosphorylation of other intracellular proteins after TCR cross-linking, JCam-1.6 cells transfected with WT LCK or 16:7:LCK-C3,5A were stimulated with the indicated antibodies and PTyr proteins immunoprecipitated and Western blotted with an anti-PTyr monoclonal anti- body (mAb). In Figure 7B, it can be seen that the pattern of PTyr bands induced by the TCR was very similar for JCam-1.6 cells transfected with either LCK construct. Thus the 16:7:LCK-C3,5A chimera appeared to facilitate the phosphorylation of all the major TCR-inducible PTyr Fig. 11. The LCK chimera can interact with ZAP-70 and, in 16:7:LCK-C3,5A-expressing cells, co-expression of WT but not C3,5A proteins. A time-course experiment also indicated that the LCK, can support TCR-induced NFAT production. Also the kinetics of TCR-induced phosphorylation of most of the submembrane topology of the 16:7:C3,5A chimera differs from that of major PTyr proteins in both cell lines were similar (Figure WT LCK. (A) Cells (3010 ) expressing WT or 16:7:C3,5A were 7C). However, the phosphorylation of a 36 kDa PTyr stimulated with OKT3 or OKT3/CD16 mAb respectively followed by protein (shown with an arrow) was found consistently to cross-linking with anti-mouse Ig, for 5 min. Following stimulation, ZAP-70 was precipitated from cell lysates using Afﬁgel-coupled be more transient in cells expressing the 16:7:LCK-C3,5A phospho-ITAM1 peptides. Immune complexes were resolved chimera compared with cells reconstituted with WT LCK. in 10% SDS–PAGE, transferred onto PVDF membrane, and Activation of the Ras-MAP kinase pathway is required co-immunoprecipitated LCK was detected with speciﬁc antibodies for induction of CD69 and NFAT by the TCR (D’Ambrosio (upper panel). The same membrane was stripped and reprobed with et al., 1994; Genot et al., 1996). To investigate the ZAP-70-reacting antibodies (lower panel). The experiment shown is representative of four experiments done with similar results. possibility that ERK MAP kinase activation might be (B) A total of 2010 16:7:LCK-C3,5A-expressing cells were affected in the 16:7:LCK-C3,5A chimera-transfected co-transfected with 15 μg of the NFAT–luciferase construct plus 25 μg JCam-1.6 cells, total cell lysates were prepared from cells of WT- or C3,5A LCK-containing plasmids as indicated. Activation of stimulated for the indicated times with anti-CD3 mAb and the cells and luciferase assays were performed as in Figure 9. The electroblotted. Blots were then probed with a phospho- experiment shown is one of four performed with identical results. Treatment of the transfected cells with ionophore and phorbol ester speciﬁc anti-ERK-1/2 antibody which recognizes the activ- induced comparable levels of NFAT-driven luciferase activity. ated, tyrosine-phosphorylated forms of ERKs 1 and 2. (C) JCam-1.6 cells (5010 ) expressing WT or 16:7:LCK-C3,5A were The blot was then stripped and reprobed with an anti- lysed in 1% Triton X-100-containing buffer and the cell lysates were ERK-1/2 antibody to detect the total amount of ERKs 1 fractionated through a sucrose gradient as described in Materials and and 2 in each lane. In Figure 10, it can be seen that methods. The content of LCK in equal aliquots from high (H) and low (L) density sucrose fractions was assessed by Western analysis. The activation of ERKs 1 and 2 was deﬁcient in the 16:7:LCK- blot was intentionally overexposed in order to detect even minimal C3,5A chimera-transfected JCam-1.6 cells compared with presence of LCK in the low density fraction. the WT LCK transfectant. These data raised the possibility that inability of the LCK chimera to reconstitute late It has been suggested that this interaction may be necessary signalling events triggered by the TCR might result from for the functional cooperation between these PTKs during its inability to support the efﬁcient activation of ERK TCR signalling (Duplay et al., 1994; Straus et al., 1996). MAP kinases. It was possible that the failure of 16:7:LCK-C3,5A to The 16:7:LCK-C3,5A chimera associates with reconstitute CD69 and NFAT expression induced by the ZAP-70 after TCR stimulation TCR in the JCam-1.6 cells might have resulted from its TCR stimulation induces LCK to associate with ZAP-70 inability to form a complex with ZAP-70. To investigate via the former protein’s SH2 domain (Duplay et al., 1994). this possibility, lysates were prepared from JCam-1.6 cells, 4991 P.S.Kabouridis, A.I.Magee and S.C.Ley expressing either WT LCK or 16:7:LCK-C3,5A, with and with GEMs (Shenoy-Scaria et al., 1993, 1994; Rodgers without mAb stimulation of their TCRs. ZAP-70 was then et al., 1994). GEMs also contain glycosylphosphatidinosi- isolated by incubating lysates with a synthetic phosphoryl- tol (GPI)-linked proteins (Mayor et al., 1994; Rodgers ated oligopeptide, corresponding to the membrane- et al., 1994) and heterotrimeric G proteins (Sargiacomo proximal ITAM of the ζ chain (Osman et al., 1995), et al., 1993). The association of LCK with both GPI- coupled to Afﬁ-Gel 10 beads (Bio-Rad). The phospho- anchored proteins and with GEMs is dependent on its ITAM peptide interacted with ZAP-70 via its SH2 domains S-acylation (Shenoy-Scaria et al., 1993; Rodgers et al., (Weiss and Littman, 1994), and, as a consequence, isolated 1994). Thus GEMs may be specialized microdomains that ZAP-70 was not bound to phospho-ITAMs of the TCR. are involved in coupling GPI-linked receptors to activation This method of puriﬁcation of ZAP-70, therefore, avoided of PTK activity. the possibility of isolating the 16:7:LCK-C3,5A chimera It was possible that the 16:7:LCK-C3,5A chimera, artefactually via its association with the TCR induced although it was targeted to the plasma membrane, might by the co-cross-linked CD3 and CD16 mAbs used for not be accessible to GEMs. To investigate this possibility, stimulating the cells. In Figure 11A, it can be seen that cell lysates were prepared from WT- and 16:7:LCK- both WT LCK and the 16:7:LCK-C3,5A chimera inducibly C3,5A-transfected JCam-1.6 cells. These lysates were associated with ZAP-70 after stimulation of the TCR. then resolved on a discontinuous sucrose gradient by Thus the failure of 16:7:LCK-C3,5A to fully complement centrifugation and low density (30/5% sucrose interface) TCR signalling in the JCam-1.6 did not result from its and high density (40% sucrose) fractions collected. GEMs failure to interact with ZAP-70. partition into the low density fraction (Brown and Rose, 1992). Fractionated lysates were then probed for LCK; as Expression of cytosolic C3,5A LCK does not expected WT LCK was clearly detected in the low density complement the signalling defect of the GEM fraction (Figure 11C). However, even after long 16:7:LCK-C3,5A chimera exposure, none of the 16:7:LCK-C3,5A chimera was The S-acyl moieties on LCK turn over with a half-life detected in the GEM fraction. These data, therefore, that is much shorter than the half-life of the protein (Paige suggested that, within the plane of the plasma membrane, et al., 1995). Deacylation may allow LCK to detach from the 16:7:LCK-C3,5A chimera was not targeted identically the membrane after activation (Milligan et al., 1995). to the WT LCK protein. Thus the failure of the 16:7:LCK-C3,5A chimera to support the induction of late activation events might have Discussion resulted from a requirement for detachment of LCK from the plasma membrane after TCR stimulation. This could This study demonstrates that S-acylation of the unique not occur when LCK was anchored artiﬁcially via a region of LCK is essential for its targeting to the plasma transmembrane domain, as was the case for 16:7:LCK- membrane. Myristoylation of LCK on its own was insufﬁ- C3,5A. cient to attach LCK ﬁrmly to the plasma membrane, as To investigate the possibility that LCK might need to revealed by analysis of the non-S-acylated C3,5A LCK detach from the plasma membrane to carry out its signall- mutant which was localized throughout the cytoplasm ing functions, 16:7:LCK-C3,5A-expressing cells were (Figure 1). This is consistent with experiments analysing transiently transfected with cDNAs encoding either WT the binding of myristoylated peptides to phospholipid or C3,5A LCK together with the NFAT luciferase reporter vesicles, which indicate that myristoylation of a protein construct. The cells were then stimulated with co-cross- cannot stably bind it to a lipid bilayer (Peitzsch and linked CD3 and CD16 mAbs, or left unstimulated, and McLaughlin, 1993; Bhatnagar and Gordon, 1997). To NFAT-driven luciferase production was assayed after 12 h achieve detectable binding of LCK to membranes, in culture. Transfection of WT LCK restored TCR induc- S-acylation was required in addition to myristoylation, as tion of NFAT in the 16:7:LCK-C3,5A-expressing JCam- indicated by the partial localization of singly S-acylated 1.6 cells. Thus the TCR signalling pathways leading to LCK to the plasma membrane. However, analysis of the NFAT production were intact in the JCam-1.6 cells which distribution of WT LCK indicated that the attachment of expressed 16:7:LCK-C3,5A. However, TCR induction of two S-acyl groups and a myristate group was necessary NFAT was not restored in cells transfected with cytosolic to achieve high levels of membrane binding. The difference C3,5A LCK (Figure 11B). These data, therefore, did not in localization of WT LCK and the two singly S-acylated suggest that the signalling defect of 16:7:LCK-C3,5A was mutants also suggests that most molecules of the WT due to its inability to detach from the plasma membrane protein carry three attached acyl groups. after TCR stimulation. In a separate series of experiments, this laboratory recently has demonstrated that the addition of the ﬁrst The 16:7:LCK-C3,5A chimera is excluded from 10 amino acids of LCK to two different soluble cyto- glycolipid-enriched microdomains plasmic proteins was sufﬁcient to retarget them to the The plasma membrane is specialized into microdomains plasma membrane and also to vesicles next to the nucleus which are enriched in glycosphingolipids, sphingomyelin in COS-7 cells (Zlatkine et al., 1997). Taken together and cholesterol but depleted of phospholipids (Brown and with the data in this study, this suggests that the SH2 and Rose, 1992; Parton and Simons, 1995). These micro- SH3 domains of LCK are not essential for intracellular domains have been termed glycolipid-enriched membranes targeting of the fully lipid-modiﬁed protein. Rather, this (GEMs) and are characterized by their insolubility in cold is achieved by dual acylation of its ﬁrst ﬁve amino acids non-ionic detergents (Rodgers et al., 1994). Some members with myristate and S-acyl moieties which directly bind of the SRC family of PTKs, including LCK, are associated LCK to the plasma membrane and perinuclear vesicles. 4992 S-acylation and signalling function of LCK LCK is associated with the co-receptors CD4 and CD8 plasma membrane. Steric hindrance by the extracellular through their cytoplasmic domains and cysteine residues domain of the 16:7:LCK-C3,5A chimera may explain why in the N-terminal unique domain of LCK, which are it was necessary to co-cross-link it with the TCR to distinct from the sites of S-acylation (Rudd, 1990). A reconstitute signalling in JCam-1.6 cells. The reconstitu- previous study demonstrated that a non-S-acylated C3S,5K tion of TCR-induced increases in intracellular free Ca mutant of LCK is not able to form a complex with CD4 in response to TCR ligation in JCam-1.6 cells was also or CD8 α when expressed in COS-7 cells, whereas singly only achieved by LCK mutants which were present at the S-acylated LCK mutants are able to complex with these plasma membrane, and the delocalized C3,5A mutant surface molecules (Turner et al., 1990). Thus, the binding was completely inactive (Figure 8A). Furthermore, the of the N-terminal unique region of LCK to CD4 or CD8 16:7:LCK-C3,5A chimera could only induce increased α correlates with the targeting of LCK to the plasma intracellular free Ca under conditions in which membrane. In addition, the COS-18 and JCam-1.6 cells phospho-ζ was formed and associated with phospho-ZAP- used in this study do not express CD4 or CD8. These 70 after co-cross-linking. Thus ζ and ZAP-70 phosphoryl- data are consistent with the hypothesis that it is the ation was closely coupled to subsequent increases in S-acylation of LCK that targets it to the plasma membrane intracellular free Ca . rather than interaction of its unique domain with cell The Sefton laboratory has also investigated the role of surface CD4 or CD8 α. lipidation in the biological activity of a constitutively One of the primary functions of LCK in TCR signalling active mutant of LCK, LCK-Y505F (Yurchak and Sefton, is to phosphorylate ITAMs in the cytoplasmic tails of the 1995). In contrast to the data from this study, a non- CD3 complex and ζ homodimers (Weiss and Littman, S-acylated C3,5S mutant of LCK-Y505F was found to be 1994; Chan and Shaw, 1996). This facilitates recruitment catalytically inactive when expressed in 208F ﬁbroblasts. of ZAP-70 PTK to the TCR, and ZAP-70 is then phos- The inactivity of this delocalized LCK-Y505F mutant phorylated by LCK and activated. Two model cell lines, correlates with its failure to be phosphorylated on Tyr394 COS-18 (Iwashima et al., 1994) and JCam-1.6 (Straus (Yurchak et al., 1996). Based on these data, this group have and Weiss, 1992), were used to study the role of LCK suggested that LCK must be associated with membranes in S-acylation in these early signalling events. Experiments order to be catalytically active, perhaps by facilitating with the heterologous COS-18 cell system indicated that Tyr394 phosphorylation. However, the present study shows phosphorylation of the CD8-ζ chimera and its association that the non-membrane-targeted C3,5A LCK mutant was with ZAP-70 occurred only with LCK mutants which highly biologically active when expressed in either COS- could localize, at least in part, to the plasma membrane 18 cells or JCam-1.6 cells. Similarly, a non-myristoylated (Figure 3). The delocalized C3,5A mutant was highly cytoplasmic form of LCK is highly active when expressed catalytically active (Figure 2B) and induced much higher in Sf9 insect cells (Carrera et al., 1991). Taken together, levels of non-speciﬁc tyrosine phosphorylation than the these data suggest that membrane attachment is only correctly localized proteins (Figure 2A), but did not required for LCK catalytic activity in certain cell types. phosphorylate CD8-ζ (Figure 3). The failure of this mutant This may reﬂect differences in the expression of either to phosphorylate the chimera was probably due to its kinases or phosphatases that act on LCK to alter its basal inaccessibility to the ITAMs of the ζ cytoplasmic tail at state of phosphorylation, thereby changing its activity. the plasma membrane. Consistent with this hypothesis, All of the LCK acylation mutants were found to be the CD16.7:LCK-C3,5A chimera, which was neither deﬁcient, to different extents, in their ability to reconstitute myristoylated nor S-acylated on LCK but which was TCR induction of two late events in JCam-1.6 cells, namely expressed efﬁciently at the plasma membrane, could CD69 and NFAT expression (Figure 5). As expected, the induce tyrosine phosphorylation of CD8-ζ when expressed C3,5A mutant, which did not reconstitute early tyrosine in COS-18 cells (Figure 6B). This suggests that one of phosphorylation stimulated by the TCR in JCam-1.6 cells, the functions of S-acylation is to target LCK to the plasma also failed to support the induction of either CD69 or membrane where it is in close proximity to the TCR. NFAT. The C3A mutant was able to support the induction Qualitatively similar results were obtained in the JCam- of CD69 and NFAT expression after TCR stimulation, but 1.6 clones in which the function of LCK acylation mutants to much lower levels than the WT protein. The C5A could be analysed in response to TCR ligation. Thus mutant was also slightly impaired in both of these late singly S-acylated LCK mutants, which were present at the responses compared with WT. The difference in function plasma membrane, were able to interact functionally with between the two singly S-acylated mutants and WT protein the TCR to induce ζ phosphorylation and its subsequent was probably due to quantitative differences in the amount association with ZAP-70 after stimulation with CD3 of LCK stably associated with the plasma membrane, antibody (Figure 4). In contrast, the delocalized C3,5A although the total amount of each mutant LCK expressed mutant completely failed to reconstitute TCR signalling. was similar to WT. This correlates well with the relative The catalytic activity of this mutant was similar to WT efﬁciency of S-acylation, which this laboratory and that LCK (Figure 2C), suggesting that its failure to complement of Lublin have found to be higher for the C5A mutant the JCam-1.6 signalling defect was due to inability to than the C3A mutant (Koegl et al., 1994; Kwong and interact with the TCR at the plasma membrane. The non- Lublin, 1995). These data, however, contrast with those acylated 16:7:LCK-C3,5A chimera was able to induce of Rodgers et al. (1994) who identiﬁed C5 as the major efﬁciently both ζ phosphorylation and its association with S-acylation site on LCK. Both of the singly S-acylated ZAP-70 after co-cross-linking of the chimera with the mutants could induce phospho-ζ and its association with TCR (Figure 7A). This again supports the hypothesis that phospho-ZAP-70 to levels comparable with WT (Figure a primary role of LCK S-acylation is to target it to the 4). The phosphorylation of the major TCR-induced 4993 P.S.Kabouridis, A.I.Magee and S.C.Ley PTyr proteins in JCam-1.6 cells transfected with the singly transfected with the chimera was reduced relative to the S-acylated mutants was also similar to WT LCK (data not WT protein (Figure 10). Since the Ras-ERK pathway is shown). The induction of CD69 and NFAT, therefore, was required for induction of CD69 and NFAT by the TCR not closely linked with the rapid tyrosine phosphorylations (D’Ambrosio et al., 1994; Genot et al., 1996), it is possible induced by TCR stimulation, and implies that LCK played that deﬁcient ERK activation by the chimera accounts an additional role in the activation process which was for its inability to support these late activation events. sensitive to its S-acylation status. However, it is possible Interestingly, anergic CD4 T cells are also deﬁcient in that the TCR-induced phosphorylation of minor PTyr their ability to activate the Ras-ERK pathway and to substrates, which were not evident when total PTyr proteins produce IL-2 following TCR stimulation (Fields et al., were analysed, was reduced with the singly S-acylated 1996; Li et al., 1996). The data in this study raise mutants, due to their inefﬁcient targeting to the plasma the possibility that an alteration of LCK function may membrane. This possibility is being investigated currently contribute to the anergic phenotype which uncouples the by two-dimensional gel electrophoresis. TCR from the efﬁcient activation of ERK MAP kinase. Yurkchak and Sefton (1995) have also investigated the Two different hypotheses may explain the partial com- importance of LCK S-acylation in T-cell function. These plementation of the JCam-1.6 signalling defect when LCK investigators tested the ability of LCK-Y505F S-acylation was artiﬁcially expressed as a transmembrane protein. mutants to induce IL-2 in stably transfected T-cell First, it was possible that LCK must detach from the hybridomas in an antigen receptor-independent fashion. plasma membrane after TCR stimulation, as a consequence These experiments failed to detect any differences between of its S-deacylation. This might be important either as a WT LCK-Y505F and singly S-acylated mutants in the mechanism to inactivate LCK or to give it access to induction of IL-2, and concluded that S-acylation of either cytosolic substrates (Milligan et al., 1995; Paige et al., Cys3 or Cys5 was sufﬁcient for full functional activity of 1995). This obviously could not occur when LCK was LCK. These data contrast with the results in this study in anchored via a transmembrane domain. However, co- which both of the singly S-acylated LCK mutants were transfection of the cytosolic C3,5A LCK mutant failed to functionally compromised relative to WT in the induction reconstitute TCR-induced NFAT production in JCam-1.6 of either NFAT or CD69 following TCR stimulation cells expressing the chimera (Figure 11B). Similarly, (Figure 5). This difference probably arises from the use an activated C3,5A LCK-Y505F mutant also did not by Sefton and colleagues of mutants which had high levels reconstitute TCR induction of NFAT in this cell line (data of constitutive activity, which were effectively uncoupled not shown). In contrast, WT LCK was able to restore from upstream regulatory events, with the result that the normal TCR signalling (Figure 11B), conﬁrming that the assay in T-hybridoma cells did not require TCR stimulation TCR signalling machinery was still intact in this cell line. to induce IL-2. In contrast, this study investigated the Taken together, these data did not support the hypothesis function of LCK acylation mutants that were not muta- that the signalling deﬁciency of the 16:7:LCK-C3,5A tionally activated in a T-cell line in which the induction chimera resulted from its inability to detach from the of IL-2 is completely dependent on TCR stimulation and plasma membrane after TCR stimulation. LCK activity. The requirements for S-acylation in LCK A second explanation to account for the signalling function, therefore, were necessarily more stringent and deﬁciency of 16:7:LCK-C3,5A was its exclusion from also more physiologically relevant. Indeed, it is not clear GEMs, in contrast to the WT protein. Thus the LCK how LCK-Y505F induces IL-2 in T hybridomas, and chimera was differentially distributed within the plane of whether this requires ζ and ZAP-70 phosphorylation. the plasma membrane relative to the WT LCK protein. Unlike the situation with the singly S-acylated LCK This may have affected the accessibility of LCK to critical mutants, the function of the LCK transmembrane chimera target proteins which were located in the GEMs. Perhaps was not limited by its level of expression at the plasma signiﬁcantly, two recent studies have suggested that Ras membrane (Figure 6C). Tyrosine phosphorylation of ZAP- is localized to caveolae, that share many properties in 70, TCR ζ and other intracellular proteins following TCR common with GEMs (Schnitzer et al., 1995; Mineo et al., stimulation was very similar between the JCam-1.6 cells 1996; Song et al., 1996). However, since lymphocytes do transfected with WT LCK or the 16:7:LCK-C3,5A chimera not contain caveolae but do contain GEMs (Fra et al., (Figure 7). The chimeric protein was also able to reconsti- 1994), the relevant plasma membrane microdomain in tute TCR-induced increases in intracellular free Ca to JCam-1.6 cells must be the latter. The exclusion of the levels greater than the WT protein (Figure 8A). Thus LCK chimera from GEMs, therefore, may prevent access retargeting of the LCK-C3,5A mutant to the plasma to critical substrates which are involved in the regulation membrane by fusing it to a transmembrane protein restored of Ras. A 36 kDa protein has been described that interacts some of its signalling functions. This is consistent with with GRB2 via its SH2 domain in T lymphocytes after the hypothesis that the primary role of S-acylation is to TCR stimulation (Buday et al., 1994; Sieh et al., 1994) target LCK to the plasma membrane where it can interact and has been postulated to couple the TCR to the activation with the TCR. However, unlike the WT protein, the of Ras. Interestingly, kinetic experiments revealed that a 16:7:LCK-C3,5A chimera was completely unable to recon- 36 kDa PTyr protein was transiently phosphorylated in stitute the induction of CD69 and NFAT by the TCR 16:7:LCK-C3,5A-expressing cells after TCR ligation, (Figure 9). The 16:7:LCK-C3,5A chimera was able to compared with a more sustained phosphorylation in cells reconstitute a sustained increase in intracellular free Ca expressing the WT protein (Figure 7C). Preliminary in the JCam-1.6 cells (Figure 8B), which is necessary for experiments have indicated that this 36 kDa PTyr protein TCR induction of IL-2 (Goldsmith and Weiss, 1988). can be precipitated with a GST–GRB2 fusion protein However, ERK MAP kinase activation in JCam-1.6 cells (data not shown). Thus it is possible that the 16:7:LCK- 4994 S-acylation and signalling function of LCK appropriate pREP3 DNA construct was then added and the cell suspension C3,5A chimera is not able to sustain prolonged phosphoryl- electroporated (Bio-Rad Gene pulser; 330 V/960 μF). The cells were ation of the GRB2-associated 36 kDa PTyr protein and, then cultured in 10 ml of complete medium for 24 h, hygromycin B as a consequence, Ras is not activated properly. This was added to a ﬁnal concentration of 0.25 mg/ml and the cells from a possibility currently is being investigated. single transfection seeded into four 96-well plates at limiting dilution (30% of wells giving rise to cell growth). Half of the medium from In conclusion, these data demonstrate an essential role each well was replaced every 3 days with fresh medium plus hygromycin for N-terminal S-acylation in the function of LCK in B, and 3 weeks later hygromycin B-resistant clones were assayed TCR signalling. This in part reﬂects a requirement for for LCK expression. Clones which expressed similar amounts of the S-acylation to target LCK to the plasma membrane where transfected LCK protein were selected for further study. it can interact with one of its critical substrates, the TCR. Antibodies However, the inability of the 16.7:LCK-C,3,5A chimera The LCK-1 anti-LCK antibody was used for confocal microscopy and to complement fully the signalling defect in the JCam-1.6 immunoprecipitations (Koegl et al., 1994). For immunoblotting, the cells indicates that the attachment of LCK to the plasma 2166 rabbit anti-LCK antibody was used, which was raised against a membrane via dual acylation of its unique domain plays denatured GST–LCK fusion protein (from Sheldon Ratnovsky, BASF, Worcester, MA). The anti-CD8 mAb, OKT8, was a kind gift from Dr some additional role which is essential for it to carry out C.Tsoukas (San Diego, CA). The anti-CD3 antibody, OKT3, was obtained its function in TCR signalling. from the American Type Culture Collection (Rockville, MD), and (Fab) fragments of this antibody were kindly prepared by A.Tutt and M.Glennie (Tenovus, Southampton, UK). Tyrosine-phosphorylated proteins were Materials and methods detected using the 4G10 anti-PTyr mAb (from Brian Druker, Oregon Health Sciences University, Portland, OR). The ZAP-4 anti-ZAP-70 Generation of LCK constructs antiserum has been described previously (Huby et al., 1995) and was A cDNA encoding wild-type mouse LCK (WT LCK) was subcloned afﬁnity puriﬁed using the immunizing peptide for immunoblotting. The neo into the KpnI–XbaI sites of the pcDNA3 expression vector (Invitrogen). TCR ζ chain was recognized in Western blots using the N39 antiserum The LCK mutants C3A, C5A and C3,5A were generated from this WT kindly provided by Jaime Sancho (Granada, Spain). The anti-CD16 mAb cDNA using PCR with the following oligonucleotides as primers: 5- and the ﬂuorescein isothiocyanate (FITC)-conjugated anti-CD69 mAb CAGGTACCATGGGCGCTGTCTGCAGCTCA-3 (C3A), 5-CAGGT- were both obtained from PharMingen. Activated ERK-1/2 was detected ACCATGGGCTGTGTCGCCAGCTCA-3 (C5A) and 5-CAGGTACC- using the PhosphoPlus p44/42 MAPK antibody from New England ATGGGCGCTGTCGCCAGCTCA-3 (C3,5A). The oligonucleotide 5- Biolabs. A rabbit antiserum which recognizes both ERKs 1 and 2 in GTCGAAGTCTCTGACCGACAG-3, containing the BamHI restriction Western blots was provided by Jeremy Tavare (University of Bristol, UK). site at position 443 of the LCK coding region, was used as the reverse primer in the PCR reaction to amplify 450 bp fragments. The various Immunoprecipitation and Western blotting analysis PCR products were digested with KpnI–BamHI restriction enzymes and Transfected COS-18 cells were washed free of serum with phosphate- then inserted into the KpnI–BamHI-treated pcDNA3/LCK construct. buffered saline (PBS) and then lysed with 1 ml of ice-cold immuno- To generate the 16:7:LCK-C3,5A chimera, the oligonucleotide 5- precipitation buffer [IPB; 150 mM NaCl, 20 mM Tris–HCl pH 7.4, 1% AAGCTTACGCGTATGGGCGCTGTCGCCAGC-3 was used as a for- NP-40, 1 mM phenylmethylsulfonyl ﬂuoride (PMSF), 100 mM Na VO , 3 4 ward primer. This oligonucleotide, which contained an MluI restriction 5 mM NaF and 5 μg/ml each of chymostatin, leupeptin and pepstatin] site 5 of the initiation ATG codon, was used for PCR in combination with scraping. JCam-1.6 cells stably expressing the various forms of the with the reverse primer described above. Following ampliﬁcation, the 7 LCK protein were resuspended to 210 cells/ml in ice-cold RPMI PCR products were digested with MluI and BamHI restriction enzymes. medium, and were left unstimulated or were stimulated for 5 min with The 16:7 fragment of the chimera was obtained from the 16:7:ZAP 1 μg/ml of OKT3 (Fab) fragments at 37°C. Cells were then pelleted chimera (Kolanus et al., 1993) following digestion with HindIII and by pulsing in a microcentrifuge and lysed in 1 ml of IPB for 15 min at MluI. The two DNA fragments were then used in a three way ligation 4°C. Cell lysates were cleared of insoluble debris by centrifugation at with a HindIII–BamHI-digested pcDNA3/LCK construct to generate the 13 000 g for 15 min at 4°C and then pre-cleared once by incubation neo 16:7:LCK-C3,5A chimera sub-cloned into the pcDNA3 expression with 10 μl of protein A–Sepharose (Pharmacia) for 15 min at 4°C. For vector. All PCR products were veriﬁed by DNA sequencing. immunoprecipitation, 10 μg of puriﬁed monoclonal antibody or 5–10 μl The various LCK mutant and chimera constructs subcloned into of antiserum were coupled covalently to 10 μl of protein A–Sepharose neo the pcDNA3 expression vector were used for transient expression with dimethylpimelimidate (Schneider et al., 1982) and incubated with experiments in COS-18 cells. To generate stable JCam-1.6 cell lines, the pre-cleared cell lysate for 4 h or overnight. Precipitation of ZAP-70 cDNA constructs were subcloned into the pREP3 episomal expression using a phospho-ITAM1 peptide coupled to Afﬁgel 10 (Bio-Rad) was vector (Hambor et al., 1988). carried out as described previously (Osman et al., 1995). Following extensive washing with ice-cold IPB, isolated protein was resolved by Cell culture and transfections SDS–PAGE and transferred onto polyvinylidene diﬂuoride (PVDF) COS-18 cell cultures were maintained in Dulbecco’s modiﬁed Eagle’s membranes. Western blotting was carried out as described previously medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM (Ley et al., 1994b). PVDF membranes were stripped of bound antibody L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The using the Amersham ECL protocol in experiments in which blots were JCam-1.6 variant of E6.1 Jurkat cells was cultured in RPMI 1640 probed for multiple antigens. medium supplemented with 5% FCS and the above concentrations of L-glutamine and antibiotics. Both cell lines were maintained in a rapid NFAT–luciferase assay growth phase prior to transfection. JCam-1.6 cells (1010 ) expressing the various LCK mutants were For transient transfections, COS-18 cells were expanded to 75% transfected by electroporation with 15 μg of pBR322-3NFAT-Luc conﬂuence in 175 cm culture ﬂasks (Nunclon) and harvested by vector (from Gerry Crabtree, Stanford, CA) using the methodology trypsinization. Detached cells were washed twice with HEPES-buffered outlined above. Cells were then cultured at 37°C for 2 h and 10 μgof saline (HBS: 140 mM NaCl, 5 mM KCl, 0.7 mM Na HPO ,20 mM OKT3 antibody added to half of the transfected cell cultures. The 2 4 HEPES pH 7.5). Approximately 510 cells, resuspended in 800 μl of remaining cultures were left unstimulated. After a further 12 h in culture, HBS, were transferred into a 0.4 cm cuvette (Bio-Rad), and the indicated cells were lysed with 150 μl of lysis buffer [0.5% NP-40, 100 mM neo amount of the appropriate pcDNA3 expression vector DNA was HK PO , 1 mM dithiothreitol (DTT) pH 7.8] for 10 min on ice. Post- 2 4 added together with sonicated salmon sperm DNA (Promega) to give a nuclear supernatants were assayed for luciferase using the Promega total of 100 μg of DNA per transfection. Cells were transfected by luciferase assay kit with a Clinilumat (Berthold) luminometer. Assays electroporation (Flowgen electroporator; 350 V, 300 μF) and then were performed in duplicate and the results shown are the mean SE. cultured for 48 h in a 10015 cm Petri dish (Nunclon) before harvesting. For the generation of stable transfectants, JCam-1.6 cells were washed Calcium analysis 6 6 twice with FCS-free RPMI 1640 medium, and 1010 cells were JCam-1.6 cells (510 ), transfected with the indicated LCK mutants, resuspended in 800 μl of the fresh serum-free medium and transferred were incubated in 1 ml of complete medium supplemented with 3 mM into an electroporation cuvette (Bio-Rad). Twenty ﬁve μg of the of the Ca indicator Indo-1 at 37°C for 1 h. Following incubation, the 4995 P.S.Kabouridis, A.I.Magee and S.C.Ley cells were washed twice with LOCKS buffer (150 mM NaCl, 1 mM microscope. Fluorescein-labelled antibody was excited at 488 nm using CaCl , 1 mM MgSO , 5 mM KCl, 10 mM glycine, 15 mM HEPES a krypton–argon mixed gas laser (Bio-Rad) and a K1 ﬁlter. Images were 2 4 pH 7.4), resuspended in the same buffer to a concentration of 110 collected using the Kalman ﬁlter settings of the COMOS programme cells/ml and then kept on ice, protected from light, until analysis. Before (Bio-Rad). Data are presented as single optical sections. analysis, the cells were pre-warmed in a 37°C waterbath for 1 min and Ca ﬂuctuations before and after the addition of the indicated ligands Cell fractionation were monitored using an LS50 Perkin Elmer Luminescence spectrometer. Transiently transfected COS-18 cells, or 1010 JCam-1.6 cells stably Cells were excited at 355 nm and emission measured at 480 and expressing LCK mutants, were resuspended in 1 ml of hypotonic buffer 405 nm, representing free versus Ca -associated Indo-1, to give an (10 mM Tris, 2 mM EDTA and 1 mg/ml each of chymostatin, leupeptin absorbance ratio. For prolonged Ca measurements, cells were loaded and pepstatin, pH 7.4), and then subjected to two successive freeze– with Indo-1 as described above with the only difference that cells were thaw cycles. The cell suspension was homogenized on ice using a washed and then cultured in a 5% FCS-containing RPMI medium. Ca Dounce homogenizer (40 strokes) and the salt concentration was then levels before and after the addition of stimulating mAbs were determined adjusted to 150 mM NaCl. Intact cells, nuclei and other debris were with a Becton Dickinson FACS Vantage. For the indicated time points, pelleted by two successive centrifugations at 480 g for 5 min. Soluble a sample of ~110 cells was analysed and the violet/blue emission and particulate fractions were generated following centrifugation at ratio was determined. When not analysed in the FACS, cell cultures 100 000 g for 30 min. Equivalent portions of the fractionated protein were were kept in a 37°C waterbath. resolved by SDS–PAGE and Western blotted with anti-LCK antibody. Flow cytometric analysis of cell surface antigens To determine cell surface expression of the 16:7:LCK-C3,5A chimera, Sucrose gradient fractionation of cell extracts immunoﬂuorescent staining was performed as described previously (Ley JCam-1.6 cells (5010 ), expressing the various LCK mutants as et al., 1994a) and cells were analysed on a Becton Dickinson FACS indicated, were washed once in PBS and then lysed for 15 min in 1 ml Vantage. To assay for the induction of CD69 expression, 110 cells of of MNE buffer (150 mM NaCl, 2 mM EDTA, 25 mM MES pH 6.5) the appropriate LCK-transfected JCam-1.6 clone were cultured in a 24- containing 1% Triton X-100 and protease inhibitors, on ice. An equal well plate (Nunclon) in the presence or absence of 10 μg/ml of the CD3 volume of an 80% sucrose solution in MNE buffer was mixed with the mAb OKT3 for 48 h. In the case of the 16:7:C3,5A chimera, stimulation lysates to form a 40% suspension, and a step sucrose gradient was was performed with CD3 plus CD16 mAbs cross-linked with the addition formed by overlaying with 2 ml of 30% sucrose–MNE and 1 ml of 5% of anti-Ig antibody. The cells were then stained with an FITC-conjugated sucrose–MNE. Isopycnic equilibriation was achieved by centrifugation CD69 mAb. Background ﬂuorescence levels were set using an FITC- at 200 000 g for 14 h in an SW55 rotor (Beckman) at 4°C. Two 2 ml conjugated mouse IgG myeloma. fractions were then collected; one from the top of the tube that contained the 30/5% sucrose interface and the other from the bottom 2 ml, which In vitro kinase assay for LCK activity contained the 40% sucrose fraction. Twenty ﬁve μl of each fraction was COS-18 cells were washed twice with PBS and then lysed in 1 ml of mixed with an equal volume of 2 Laemmli sample buffer and the of ice cold buffer (SB) comprising 50 mM N-octyl-β-D-glucopyranoside proteins were separated by 12% SDS–PAGE, transferred onto PVDF (Sigma), 150 mM NaCl, 25 mM Tris, 1 mM Na VO , 2 mM Na P O , membrane and then probed with anti-LCK antibody. 3 4 4 2 7 20 mM NaF and 1 μg/ml each of chymostatin, leupeptin and pepstatin, pH 7.2. Cell lysates were cleared of insoluble material by centrifugation at 13 000 g for 10 min. Precipitation with anti-LCK antibody coupled Acknowledgements to protein A–Sepharose for 4 h was performed as described above. Immunoprecipitates were then washed ﬁve times in lysis buffer and The authors would like to thank A.Weiss, G.Crabtree, M.Tykocinski, once in kinase buffer (100 mM NaCl, 50 mM HEPES pH 7.5, 5 mM S.Ratnovsky and D.Littman for the reagents used in this study. The MgCl , 5 mM MnCl and1mM Na VO ). Each immunoprecipitate was 2 2 3 4 authors are also grateful to the Photo-Graphics department at NIMR and then resuspended in 20 μl of kinase buffer supplemented with 10 μM to E.Hirst for their help with the ﬁgures. This work was supported by ATP, 5 μCi [ P]ATP and 1 mM RR-SRC peptide substrate (Gibco- the Medical Research Council. BRL) and incubated at room temperature for 30 min. The reaction was stopped by addition of 30 μl of kinase buffer plus 25 mM EDTA and then peptide phosphorylation was measured by binding to p81 paper (Whatman) and Cerenkov counting. References To assay the activity of LCK expressed in the JCam-1.6 clones, cells Appleby,M.W., Gross,J.A., Cooke,M.P., Levin,S.D., Qian,X. and were lysed using SB containing 1% Triton X-100 instead of N-octyl-β- Perlmutter,R.M. (1992) Defective T cell receptor signaling in mice D-glucopyranoside. Immunoprecipitation was carried out as for COS-18 fyn lacking the thymic isoform of p59 . 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The EMBO Journal – Springer Journals

Published: Aug 15, 1997

Keywords: LCK; localization; S‐acylation; signalling

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S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

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S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

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