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Phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3)/Tec kinase‐dependent calcium signaling pathway: a target for SHIP‐mediated inhibitory signals

Phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3)/Tec kinase‐dependent calcium signaling... The EMBO Journal vol.17 no.7 pp.1961–1972, 1998 Phosphatidylinositol-3,4,5-trisphosphate (PtdIns- 3,4,5-P )/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals with the ability to interact with PtdIns-3,4,5-P or PtdIns- Andrew M.Scharenberg, Ousama El-Hillal, 3,4-P in vitro have now been described (Rameh et al., David A.Fruman , Laurie O.Beitz, 2 2 3,4 1995, 1997; Klarlund et al., 1997). Recently, the task of Zuomei Li , Siqi Lin, Ivan Gout , 1 5 determining whether and/or how each of these in vitro Lewis C.Cantley , David J.Rawlings and interactions translate into cellular functions was begun by Jean-Pierre Kinet three different groups who provided evidence that PtdIns- Laboratory of Allergy and Immunology and Laboratory of Signal 3,4-P and PtdIns-3,4,5-P act as upstream activation 2 3 Transduction, Beth Israel Deaconess Medical Center and Harvard signals for the AKT serine/threonine kinase via an inter- Medical School, Boston, MA 02215, Department of Microbiology action with its pleckstrin homology (PH) domain (Franke and Molecular Genetics, University of California at Los Angeles, 5 et al., 1995, 1997; Alessi et al., 1997; Klippel et al., 1997; Los Angeles, CA 90095-1662, Department of Pediatrics, University Stokoe et al., 1997). The cellular functions attributable to of California at Los Angeles, Los Angeles, CA 90095-1752, USA, Ludwig Institute for Cancer Research, 91 Riding House Street, other in vitro protein–PtdIns-3,4,5-P /PtdIns-3,4-P inter- 3 2 London W1P 8BT, UK and Institute of Molecular Biology and actions remain uncharacterized, but the diversity of pro- Genetics, Kyiv, Ukraine teins which carry these domains suggests that PtdIns- Corresponding author 3,4,5-P /PtdIns-3,4-P are likely to act in an analogously 3 2 e-mail: [email protected] diverse range of processes depending on the cellular context in which they are produced. Tec family non-receptor tyrosine kinases have been The PH domain of Bruton’s tyrosine kinase (Btk), a implicated in signal transduction events initiated by member of the Tec non-receptor tyrosine kinase family, cell surface receptors from a broad range of cell types, is among the protein domains capable of interacting with including an essential role in B-cell development. A PtdIns-3,4,5-P in vitro (Salim et al., 1996; Rameh et al., unique feature of several Tec members among known 1997). Tec kinases are an emerging family of proteins tyrosine kinases is the presence of an N-terminal which are expressed in both hematopoietic and non- pleckstrin homology (PH) domain. We directly demon- hematopoietic tissues. Four Tec members (Tec, Btk, Itk strate that phosphatidylinositol-3,4,5-trisphosphate and Bmx) have closely homologous structures which (PtdIns-3,4,5-P ) interacting with the PH domain acts include an N-terminal PH domain, followed by SH3, SH2 as an upstream activation signal for Tec kinases, and tyrosine kinase domains (reviewed in Desiderio and resulting in Tec kinase-dependent phospholipase Cγ Siliciano, 1994; Rawlings and Witte, 1995). These Tec (PLCγ) tyrosine phosphorylation and inositol tris- kinases have been implicated in the signaling pathways phosphate production. In addition, we show that this of a variety of hematopoietic receptors, including several pathway is blocked when an SH2-containing inositol types of cytokine and antigen receptors (reviewed in phosphatase (SHIP)-dependent inhibitory receptor is Rawlings and Witte, 1995). The mechanism of their engaged. Together, our results suggest a general mech- activation by these receptors is thought to involve a two- anism whereby PtdIns-3,4,5-P regulates receptor- step process in which they receive a currently undefined dependent calcium signals through the function of signal which targets them to the vicinity of activated Src Tec kinases. kinases, after which their activation occurs through Keywords: B-cells/inositol trisphosphate/phospholipase a transphosphorylation/autophosphorylation mechanism C/receptor/tyrosine kinases (Mahajan et al., 1995; Rawlings et al., 1996). In terms of downstream targets, Btk is thought to play an important role in apoptotic signaling as well as the activation of Jnk kinases and phospholipase Cγ2 (PLCγ2) (Rigley et al., Introduction 1989; Takata and Kurosaki, 1996; Uckun et al., 1996; Kawakami et al., 1997). Furthermore, B-cell overexpres- Receptors of virtually every type have been shown to sion experiments show that participation in PLCγ2 activa- stimulate the production of the phosphoinositides phos- tion is a general property of Tec kinases, that their role phatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P ) and in PLCγ2 activation is distinct from that of Src or Syk PtdIns-3,4-P (Auger et al., 1989; Serunian et al., 1991; family kinases, and that they are particularly important Gold and Aebersold, 1994; Gold et al., 1994; Okada et al., for producing the sustained production of inositol tris- 1996). These lipids are produced by the action of a family phosphate (IP ) required for store-operated calcium influx of enzymes which specifically phosphorylate the D3 (Fluckiger et al., 1998). position of the inositol ring in phosphoinositides (reviewed While progress has been made in identifying signaling in Carpenter and Cantley, 1996; Vanhaesebroeck et al., 1997), and are thought to act as inducible membrane pathways lying upstream and downstream from Tec kin- targeting signals for proteins which carry domains capable ases, little is known about Tec kinase structure–function of interacting with them. Several examples of domains relationships. Many investigators have attempted to under- © Oxford University Press 1961 A.M.Scharenberg et al. stand Tec signaling mechanisms by identifying ligands for for their ability to transform rat fibroblasts synergistically the subdomains of these kinases. Four potential cellular with Btk. This screen involves assaying the transforming ligands for Tec family PH domains have emerged from activity of Btk alone or in combination with other molec- in vitro studies including: (i) G-protein β/γ subunits ules using retroviral expression of the proteins of interest (Tsukada et al., 1994); (ii) protein kinase C (most isoforms) in a soft agar growth transformation assay, and has (Yao et al., 1994; Kawakami et al., 1995); (iii) BAP-135, previously permitted receptor-independent analysis of Btk a 135 kDa protein of unknown function (Yang and activation and signaling (Afar et al., 1996). Using this Desiderio, 1997); and (iv) inositol-1,3,4,5-tetrakisphos- assay, Btk or an activated form of p110, known as p110* phate (IP , a soluble inositol phosphate) and PtdIns-3,4,5- (Hu et al., 1995), each produce 10 colonies per plate, P (Fukuda et al., 1996; Salim et al., 1996; Rameh et al., while their co-expression results in an ~6-fold enhance- 1997). Several proteins, including CBL, WASP and SAM- ment in number of colonies. In contrast, co-expression of 68 among others, have been proposed to function in Tec Btk with Cbl or Vav, both of which have also been shown family signaling via their ability to interact with Tec to interact with Btk in vitro, does not produce a similar family SH3 domains in vitro (Cory et al., 1995; Bunnell enhancement (Z.Li, D.J.Rawlings, H.Park and O.N.Witte, et al., 1996; Andreotti et al., 1997). Finally, Tec has been unpublished data). proposed to function in linking cytokine receptors to Vav (through a direct association) and phosphatidylinositol 3- p110–Tec kinase co-expression synergistically kinase (PI3K)-dependent signaling pathways (through an enhances BCR-mediated IP production and association with the p85 subunit of PI3K) based on co- calcium signals precipitation and in vitro binding data (Machide et al., Increased expression of Tec kinases in B cells produces 1995; Takahashi-Tezuka et al., 1997). It is currently an enhanced calcium signal characterized in particular by unclear whether and/or how each of the binding inter- sustained calcium influx (Fluckiger et al., 1998). Based actions described for various Tec family subdomains are on the results of the transformation screen, we investigated integrated into the signaling function of these kinases. whether the p110 subunit of PI3K could functionally However, compelling evidence for the relevance of PH interact with Tec kinases during calcium signaling domain function to Tec kinase function exists in the form (Figure 1A). In a typical experiment, p110 expression of spontaneously arising mutations in the Btk PH domain produces only a minor change in the overall calcium flux which cause X-linked agammaglobulinemia in humans relative to control infections, while Btk, Itk and Tec and mice (reviewed in Conley and Rohrer, 1995; Rawlings expression produce an enhanced calcium signal most and Witte, 1995). Furthermore, one such mutation, an evident at later time points. However, when expressed arginine to cysteine substitution at position 28, has been together with p110, the effects of Btk, Itk and Tec shown to reduce the in vitro inositol polyphosphate- and are substantially larger and more sustained—reproducibly phosphoinositide-binding activity of the isolated Btk PH greater than the additive effects of p110 and the kinase domain (Fukuda et al., 1996; Salim et al., 1996; Rameh alone. Blots of the lysate (data not shown) demonstrate et al., 1997). that similar amounts of each kinase are expressed when Given the confusing picture of Tec kinase function Btk, Itk and Tec are expressed alone or with p110. which has emerged from studying the interactions of its In order to test whether the PI3K activity of p110 and various domains in vitro, we undertook a functional not some other factor related to its increased expression approach to studying Tec kinase signaling by examining level was responsible for the observed synergistic effects how co-expressing them with other signaling proteins on the calcium signal, we examined the effect of wortman- affects Tec kinase-regulated signaling events in vivo. This nin on the calcium flux generated by expression of Tec approach identified an activated form of the p110 subunit kinases alone or in combination with p110 (Figure 1B, of PI3K (p110) as a prominent synergizing molecule when note that the traces in this experiment were obtained from expressed with the Tec kinase prototype Btk in a fibroblast the same experiment as the Btk traces without wortmannin transformation assay. Subsequent expression of Btk, Itk pre-treatment in Figure 1A). As expected and as previously and Tec with p110 in B cells showed a similar functional reported, wortmannin inhibits the calcium fluxes of unin- synergy at the level of the B-cell receptor (BCR)-initiated fected A20 B cells and of A20 B cells infected with a calcium signal, and this system was used for a detailed control recombinant vaccinia virus (Kiener et al., 1997; molecular analysis of the synergy mechanism. The results A.M.Scharenberg and J.-P.Kinet, unpublished data; of this analysis suggest that PtdIns-3,4,5-P initiates Figure 1B). Wortmannin is also able to reverse the minor PLCγ2-dependent IP production at least in part through enhancement of calcium flux seen with infection with the its ability to interact with and activate Tec kinases. In p110 virus alone, indicating that the effect of p110 on addition, we show that FcγRIIb1, an inhibitory receptor calcium mobilization is mediated through its phosphoinosi- which recruits the SHIP inositol 5-phosphatase (Ono tide metabolites. In addition, wortmannin inhibits essen- et al., 1996), eliminates BCR-induced PtdIns-3,4,5-P tially all of the calcium flux enhancement generated by accumulation and results in blocked Tec kinase-dependent Btk expression alone, and substantially but incompletely calcium signaling. inhibits the effect of p110–Btk co-expression. The com- plete inhibition of the effect of Btk expressed alone has Results the important implication that the action of endogenous p110-PI3K synergizes with Btk in a transformation PI3K is required for Btk to function in calcium signaling. assay The less complete inhibition of calcium mobilization in In order to identify signaling proteins which functionally Btk–p110-expressing cells is in part expected, as the interact with Tec kinases, proteins of interest were screened amount of wortmannin used would produce less complete 1962 PIP /Tec kinase calcium signaling pathway Fig. 1. p110–Tec kinase co-expression synergistically enhances BCR-mediated IP production and calcium signals. (A) p110–Tec kinase co- expression produces synergistically increased calcium signals. Upper panels: A20 B cells were plated to 20% confluency and subsequently infected for 13 h with the indicated combinations of recombinant vaccinia viruses. The following morning, the cells were harvested, loaded with fura-2, and calcium mobilization in response to 15 μg/ml Fab rabbit anti-mouse IgG was measured in a bulk spectrofluorimeter at 30°C. (B) Btk-dependent enhancement of calcium signaling is inhibitable by wortmannin. The same experiment as (A), except that cells were pre-treated with 100 nM wortmannin for 10 min prior to the assay. (C) p110–Btk co-expression produces a synergistically increased IP signal. Cells were infected and harvested identically to those in (A). After harvest, they were suspended at 510 cells/ml in calcium buffer and stimulated with 30 μg/ml of Fab rabbit anti-mouse IgG. After the indicated times of stimulation, cells were spun down rapidly and lysed in 20% trichloroacetic acid (w/v in H O), and prepared for IP radioreceptor assay as specified in the manufacturer’s protocol. inactivation of PI3K activity in these cells because its examined whether p110 would be able to complement the pharmacologic target, the p110 PI3K subunit, is present signaling capabilities of mutant versions of Tec kinases. in greater abundance. Similar results are obtained with Because of the availability of a variety of Btk mutants in other Tec kinases in the A20 cells (data not shown). our laboratory, Btk was again used as the prototype Tec Tec kinase-dependent enhancement of calcium signaling kinase for these experiments. Btk constructs carrying occurs at least in part via enhanced production of IP inactivating mutations within each major domain [kinase (Fluckiger et al., 1998). Using Btk as a prototype, we (K430R), PH-R28C, ΔSH3 (full deletion) and SH2-R307K evaluated whether increased Tec kinase expression in (Figure 2A–D)] were expressed in A20 B cells, and combination with p110 was affecting IP production calcium flux (left panel) and IP generation (right panel) 3 3 (Figure 1C). Control infected A20 cells produce an ~2- were analyzed for expression of each mutant kinase alone fold increase in IP at 1 min after BCR engagement, or with p110 (designated black traces). Traces representing which then decays back to just above baseline by 10 min. control and p110 alone infections for each experiment are While expression of p110 alone increases IP production shown in gray for purposes of comparison. As shown by only slightly relative to control, Btk expression alone the designated black traces in Figure 2A, the catalytically produces a substantial enhancement, and p110–Btk co- inactive mutant expressed alone is not able to enhance expression results in synergistically higher and more the calcium (left panel) or IP signals (right panel) sustained IP production than Btk alone. Therefore, the significantly, and co-expression with p110 is not able to synergy between p110 and Tec kinases observed at the complement its loss of signaling function. Of the Btk level of the calcium signal correlates well with the interaction domain mutants, the Btk-ΔSH3 produces both production of IP , suggesting that p110 and Tec kinases enhancement of signaling when expressed alone and are both somehow involved in the regulation of PLCγ2, synergistic signaling when co-expressed with p110 the principal PLC isozyme utilized during BCR signaling. (Figure 2C, direct comparison of the ΔSH3 and wild-type Btk in other experiments showed that the ΔSH3 mutant p110–Tec synergy requires intact PH, SH2 and reproducibly generated similar but somewhat larger effects kinase domains than wild-type Btk, data not shown). The R28C (Figure 2B) In order to begin to analyze the mechanism through which and R307K (Figure 2D) mutants were unable to enhance p110 and Tec kinases were acting on IP production, we calcium signaling nor did they show significant comple- 1963 A.M.Scharenberg et al. effects of activation of other kinases, the direct products of p110-PI3K action are known—PtdIns-3,4,5-P and PtdIns-3,4-P . Therefore we decided to begin determining which of the above scenarios best explained the p110– Tec synergy by testing the hypothesis that Tec kinases were enhancing p110 signaling. One implication of the hypothesis that Tec kinases act upstream from p110 would be that production of the direct p110 product would be increased. Available evidence suggests that PtdIns-3,4,5-P is the primary direct product generated by p110 (via 3 phosphorylation of PtdIns-4,5- P ) while PtdIns-3,4-P is generated indirectly via 5 2 2 dephosphorylation of PtdIns-3,4,5-P (Stephens et al., 1991). Therefore, we first examined the effect of co- expression of p110 and Btk on the basal and stimulated levels of PtdIns-3,4,5-P in A20 B cells (Figure 3A). In control cells, PtdIns-3,4,5-P is just detectable in our TLC assay after 2 min of stimulation, consistent with what has been previously published on PtdIns-3,4,5-P production in tumor B-cell lines (Gold and Aebersold, 1994). This production is enhanced significantly by expression of p110 alone, and increased marginally by expression of Btk alone at these expression levels (at higher expression levels achieved by longer infections, significant elevations in PtdIns-3,4,5-P are detectable with Btk expression alone; A.M.Scharenberg, O.El-Hillal and J.-P.Kinet, unpublished observations; see also Figure 3C below). However, co- expression of Btk and p110 results in much higher levels of PtdIns-3,4,5-P than p110 alone or Btk alone in both basal and stimulated conditions, indicating the presence of a p110–Tec kinase-dependent synergy on the level of cellular PI3K lipid products. As substitution of other Tec kinases in these experiments produced identical synergy effects (data not shown), we focused on Btk for subsequent experiments because of the ready availability of Btk Fig. 2. p110–Tec synergy requires intact Btk PH, SH2 and kinase mutants and immunological reagents in our laboratory. domains. (A–D) For calcium assays, A20 B cells were infected with A second implication of the hypothesis that Tec kinases the indicated vaccinia viruses, harvested, loaded with fura-2, act upstream from p110 is that loss-of-function Tec mutants stimulated and assayed in a manner identical to those in Figure 1. For would be expected to show a loss of the synergy at the IP assays, A20 B cells were infected with the indicated vaccinia viruses, harvested, stimulated, lysed in 20% TCA, and processed for level of PtdIns-3,4,5-P . Therefore, we compared the IP assay in a manner identical to those in Figure 1. Note that for both PtdIns-3,4,5-P signal generated by co-expression of p110 the calcium and IP assays, the K430R, R28C and R307K mutants and wild-type Btk with that generated by co-expression were assayed in the same experiment, so that their control and p110 of p110 and inactive Btk (Btk-K430R, which is unable to alone curves (shown in gray) are identical. The ΔSH3 was assayed in calcium signal and is not complemented by p110, see a separate experiment, so the control curves from that particular experiment are shown for comparison. Figure 2) (Figure 3B, left panel). Similar experiments were performed in NIH 3T3 fibroblasts in the absence of mentation of their signaling defects when expressed with receptor stimulation (right panel). These experiments show p110. that the inactive Btk mutant has equivalent or greater ability than wild-type Btk to enhance the PtdIns-3,4,5-P p110–Tec co-expression synergistically enhances signal. They also demonstrate that no hematopoietic- detectable PtdIns-3,4,5-P specific component is required for the synergistic PtdIns- Based on published reports of ligands of the various 3,4,5-P signal. domains of Tec kinases (see Introduction) and the recent Again assuming that Tec kinases act upstream from suggestion that p110 might be a cofactor for PLCγ p110, the ability of inactive Btk to produce synergistic activation (Rhee and Bae, 1997), the p110–Tec kinase accumulation of PtdIns-3,4,5-P when co-expressed with synergy could reasonably be explained by four possible p110 could only be accounted for if p110 activation was mechanisms: (i) p110-dependent enhancement of Tec being enhanced by Btk in a manner which was independent kinase signaling; (ii) Tec kinase-dependent enhancement of Btk activity. In order to address this possibility, we used of p110 signaling; (iii) a combination of both of these HPLC to analyze the effect of increased Btk expression on mechanisms; or (iv) a cooperative mechanism in which both PtdIns-3,4,5-P and PtdIns-3,4-P (Figure 3C, left 3 2 Tec kinases and p110 act independently on PLCγ2 function. and right panels). Increased Btk expression is associated While identifying direct targets of protein kinases can be with a large (~3-fold) increase in PtdIns-3,4,5-P levels difficult in a cellular context due to the confounding with essentially no effect on PtdIns-3,4-P levels. Since 1964 PIP /Tec kinase calcium signaling pathway Fig. 3. p110–Tec co-expression synergistically enhances detectable PtdIns-3,4,5-P .(A) p110–Btk co-expression produces a synergistic enhancement of the amount of detectable PtdIns-3,4,5-P (PIP3). Both panels: A20 B cells were infected for 13 h with the indicated vaccinia viruses, harvested, labeled with P and stimulated or not for 2 min with 30 μg/ml Fab rabbit anti-mouse IgG. Lipids were then extracted and analyzed by TLC. (B) Btk kinase activity is not required to produce an enhanced PtdIns-3,4,5-P signal. Left panel: A20 B cells were infected with the indicated vaccinia viruses, and then analyzed for PtdIns-3,4,5-P levels as in (A). Right panel: NIH 3T3 fibroblasts were infected for 6 h with the indicated vaccinia viruses, harvested and labeled with P. Lipids were then extracted and analyzed by TLC. (C) Increased Btk expression does not significantly affect production of PtdIns-3,4-P . A20 B cells were infected for 16 h with Btk alone or control vaccinia viruses. Cells were then harvested, labeled with P, stimulated or not for 2 min with 30 μglml Fab rabbit anti-mouse IgG and lipids were extracted and analyzed by HPLC. Note that expression when using a single virus is typically greater than when co-infections are performed, so that Btk expression in these experiments was in the range of 2-fold greater than in all other experiments. (D) An intact PH domain but not SH2 domain is required to produce synergistic enhancement of PtdIns-3,4,5-P . Left panel: A20 B cells were infected for 13 h with the indicated vaccinia viruses, and then analyzed for PtdIns-3,4,5-P levels as in (A). Right panel: NIH 3T3 fibroblasts were infected for 6 h with the indicated vaccinia viruses, and then otherwise processed as in (B). p110 activation typically produces proportional accumula- The remaining explanation for the ability of increased Tec tion of both PtdIns-3,4,5-P and PtdIns-3,4-P (Hu et al., kinase expression to increase the accumulation of PtdIns- 3 2 1995; Klippel et al., 1996), the selective enhancement in 3,4,5-P was that it was somehow reducing the rate of PtdIns-3,4,5-P levels suggests that Btk is not significantly PtdIns-3,4,5-P degradation. Since Btk is known to be 3 3 affecting cellular PI3K activity. capable of binding PtdIns-3,4,5-P via its PH domain, Based on the above two experiments, we felt that it the simplest explanation for how increased Tec kinase was unlikely that Tec kinases were acting upstream to expression could decrease PtdIns-3,4,5-P degradation was activate p110 and enhance D3 phosphoinositide synthesis. by binding to PtdIns-3,4,5-P and thereby protecting it 1965 A.M.Scharenberg et al. from endogenous inositol phosphatases. We therefore tested which interaction domains of Btk were participating in the enhancement of PtdIns-3,4,5-P accumulation by expressing the R28C and R307K Btk mutants alone or with p110-PI3K in A20 B cells (Figure 3D, left panel). The R28C PH domain mutation (which greatly reduces the binding of the isolated Btk PH domain to PtdIns- 3,4,5-P in vitro) eliminates the enhancement of the PtdIns-3,4,5-P signal seen after co-expression of p110. In contrast, an enhanced PtdIns-3,4,5-P signal identical to that of wild-type Btk is seen for the R307K SH2 FLVR mutant. A comparable result is obtained if the co- expression is performed in fibroblasts (right panel), again confirming that additional hematopoietic-specific compon- ents are not required. This experiment also shows that the ability of the ΔSH3 mutant to enhance PtdIns-3,4,5-P detection is comparable with that of other forms of Btk (right panel), consistent with its containing an intact PH domain and its ability to produce enhanced calcium signaling. Finally, together with the lack of significant enhancement of PtdIns-3,4-P accumulation in the HPLC experiment in Figure 3B above, this experiment provides in vivo data to support the previously described binding specificity of the Btk PH domain for PtdIns-3,4,5-P . Btk-dependent PLCγ2 tyrosine phosphorylation is PI3K-dependent The above data demonstrate that Tec kinase PH domains are interacting with PtdIns-3,4,5-P in the context of our co-expression system, and that Tec kinases do not act as major upstream activation signals for p110. They therefore constrain the potential mechanisms for the functional synergy between Tec kinases and p110 to those in which PtdIns-3,4,5-P acts either upstream from or together with the Tec kinase to promote IP production and calcium mobilization through the activation of PLCγ2. Since Btk is known to participate in the tyrosine phosphorylation of PLCγ2, we first evaluated whether the synergistic effect on PLCγ2 activation (as assessed by IP Fig. 4. Btk-dependent PLCγ2 tyrosine phosphorylation is PI3K- production) could be accounted for via synergistic effects dependent. (A) BCR-mediated PLCγ2 tyrosine phosphorylation is on PLCγ2 tyrosine phosphorylation. Using conditions enhanced by p110–Btk co-expression. A20 B cells were infected for identical to those used in the calcium synergy and IP 13 h with the indicated vaccinia viruses, harvested, washed once with calcium buffer, resuspended at 10 cells/ml in calcium buffer and experiments, we expressed various combinations of p110 stimulated or not for 2 min with 30 μg/ml Fab rabbit anti-mouse and Btk in the A20 B-cell line, and then analyzed the IgG. Cells were then quickly spun down and lysed, and post-nuclear level of BCR-induced PLCγ2 tyrosine phosphorylation supernatants were immunoprecipitated with the indicated antibodies or (Figure 4A). Relative to control infection, expression of directly analyzed. (B) p110–Btk-dependent PLCγ2 tyrosine phosphorylation requires kinase activity and intact PH and SH2 p110 alone produces a slight enhancement of PLCγ2 domains. A20 B cells were infected for 13 h with the indicated tyrosine phosphorylation, consistent with the idea that vaccinia viruses, and otherwise processed identically to those in (A). p110 is able to functionally interact with endogenous Btk. (C) Wortmannin treatment of A20 B cells overexpressing Btk or In contrast, Btk expression alone produces an easily p110–Btk inhibits PLCγ2 tyrosine phosphorylation. A20 B cells were detectable enhancement, which is then enhanced further infected for 13 h with the indicated vaccinia viruses, and otherwise processed identically to those in (A), except that the indicated samples by co-expression with p110. were pre-treated for 10 min with 100 nM wortmannin. If enhanced PLCγ2 tyrosine phosphorylation is respons- ible for the functional synergy between p110 and Btk, then PLCγ2 tyrosine phosphorylation should be eliminated of p110 to complement them in calcium signaling and by Btk mutants which eliminate the synergy seen at the IP assays. levels of IP and calcium. We tested this by expressing As a further test of the hypothesis that modulation of p110 alone or with wild-type or loss-of-function Btk PLCγ2 tyrosine phosphorylation by p110–Btk accounted mutants and analyzing BCR-induced PLCγ2 tyrosine for the synergistic enhancement of calcium signaling, we phosphorylation as above (Figure 4B). All loss-of-function treated cells overexpressing Btk or p110–Btk with 100 nM Btk mutations greatly reduce the enhancement of PLCγ2 wortmannin for 10 min and analyzed PLCγ2 tyrosine tyrosine phosphorylation produced by co-expression of phosphorylation (Figure 4C). Consistent with the effect p110 and wild-type Btk, consistent with the inability of wortmannin on the calcium signal (see Figure 1 1966 PIP /Tec kinase calcium signaling pathway above), PLCγ2 tyrosine phosphorylation is inhibited by In order to analyze the mechanism of p110-induced Btk wortmannin pre-treatment in both Btk- and p110–Btk- autophosphorylation, we examined the effect of the various overexpressing cells. The greater residual level of PLCγ2 Btk interaction domain mutations on the ability of p110 tyrosine phosphorylation in p110–Btk-overexpressing cells to induce Btk autophosphorylation (Figure 5B) in A20 B- than in cells expressing Btk alone may partially explain cell and fibroblast environments. The R28C PH domain why wortmannin does not completely revert the effect mutant eliminates p110-dependent Btk tyrosine phospho- of p110–Btk co-expression on the calcium signal (see rylation in both environments. In comparison, the level of Figure 1B above). Finally, the ability of wortmannin to tyrosine phosphorylation of the ΔSH3 mutant is similar attenuate the enhancement of PLCγ2 tyrosine phosphoryla- to that of wild-type Btk, and that of the R307K mutant is tion produced by Btk expression alone has the important markedly enhanced. Together with the results from Figures implication that Btk-dependent tyrosine phosphorylation 1–4, these results suggest that PtdIns-3,4,5-P acts as an of PLCγ2 is normally a PI3K-dependent process. upstream activation signal for Btk through an interaction with the Btk PH domain, and that the SH3 and SH2 PtdIns-3,4,5-P interaction with the Btk PH domain domains are dispensable for this process. It is important induces Btk autophosphorylation to note that the ΔSH3 mutant is missing one phosphoryla- The synergistic effect of p110–Btk co-expression on tion site at residue 223, so its phosphorylation level would PLCγ2 tyrosine phosphorylation suggests that co-expres- normally be expected to be ~50% of wild-type Btk (Park sion of Btk and p110 is either directly affecting Btk et al., 1996). The enhanced autophosphorylation observed activation or is somehow co-localizing Btk and PLCγ2, for the R307K mutant was unexpected, but we speculate with either scenario resulting in enhanced Btk-dependent that since the Btk SH2 domain is required for Btk- tyrosine phosphorylation of PLCγ2. We and others have dependent phosphorylation of PLCγ2, this mutation might shown previously that Btk activation, which is identical somehow inhibit access of Btk to substrates, thereby to that seen after BCR stimulation, can be induced by co- enhancing its opportunities for autophosphorylation. expression of Btk with Src kinases, and that this occurs by enhanced autophosphorylation of Btk (Mahajan et al., The FcγRIIB1–SHIP inhibitory receptor complex 1995; Rawlings et al., 1996). Since enhanced Btk activa- blocks PtdIns-3,4,5-P /Tec kinase-dependent tion would explain the enhanced PLCγ2 tyrosine phospho- calcium signaling rylation observed with p110–Btk co-expression, our initial The inhibitory receptor FcγRIIb1 is expressed on B investigations focused on whether p110 could be involved cells, where its co-engagement with the BCR results in in promoting Src kinase-induced Btk autophosphorylation. an inhibitory signal which is thought to provide an We first examined whether p110 alone was able to important negative feedback mechanism for antibody induce Btk autophosphorylation using the fibroblast co- production. The FcγRIIb1 inhibitory signal is known to expression system in which our previous studies were cause a block in sustained calcium signaling due to its performed (Figure 5A, top left panel). p110 induces an apparent ability to specifically block calcium influx easily detectable enhancement of wild-type Btk tyrosine (Bijsterbosch and Klaus, 1985; Amigorena et al., 1992; phosphorylation which is entirely dependent on Btk activ- Muta et al., 1994), and recently has been shown to ity. We then investigated whether Src kinases could require the SHIP inositol phosphatase, an enzyme with synergize with p110 in inducing Btk phosphorylation the in vitro capability to degrade PtdIns-3,4,5-P (Gupta (Figure 5A, top right panel) and whether Src kinase- et al., 1997; Ono et al., 1997). Since PtdIns-3,4,5-P / induced Btk phosphorylation was wortmannin inhibitable Tec kinase-dependent calcium signals are sustained as (bottom left panel). Co-expression of Lyn and Btk or of the result of enhanced calcium influx (Fluckiger et al., p110 and Btk both induce Btk tyrosine phosphorylation, 1998), we examined how co-engagement of the BCR and expression of all three produces a further enhancement and FcγRIIb1 affected PtdIns-3,4,5-P levels and Tec of this. In addition, treatment of cells expressing Lyn and kinase-dependent signaling (Figure 6). We first compared Btk with 100 nM wortmannin substantially inhibits the the levels of PtdIns-3,4,5-P observed after either BCR Lyn-dependent Btk tyrosine phosphorylation. Alterations engagement alone or co-engagement with FcγRIIb1 in Lyn activity (as assessed by its ability to induce tyrosine (Figure 6A). While BCR engagement resulted in a phosphorylation of the Syk tyrosine kinase under identical sustained PtdIns-3,4,5-P signal, no PtdIns-3,4,5-P was 3 3 conditions) by wortmannin or p110 expression do not detectable in this experiment at any time point after BCR– occur (data not shown). In further support of the idea that FcγRIIb1 co-engagement, although in other experiments p110 acts in concert with Src kinases, p110γ produces a small amounts of PtdIns-3,4,5-P could be detected at similar enhancement of Btk tyrosine phosphorylation only early time points only (data not shown). We then when it is co-expressed with Btk in a rat fibroblast line examined the effect of BCR–FcγRIIb1 co-engagement which constitutively expresses an activated Src mutant on Tec kinase-dependent calcium signaling and PLCγ2 (srcE387G) (Li et al., 1997). Identical p110-dependent tyrosine phosphorylation. In both control and Btk- enhancement of Btk tyrosine phosphorylation also occurs overexpressing B cells, FcγRIIb1 was able to inhibit in the A20 B-cell environment when Btk and p110 are sustained calcium signals (Figure 6B) and Btk-dependent co-expressed (Figure 5A, bottom right panel), and is PLCγ2 tyrosine phosphorylation (Figure 6C). Similar wortmannin inhibitable (data not shown). These results results for the calcium signal were obtained when other closely parallel the observed Btk-dependent PLCγ2 tyros- Tec kinases were expressed (data not shown). Strikingly, ine phosphorylation and IP production, consistent with analysis of Btk tyrosine phosphorylation in both control our previous findings that Btk autophosphorylation is and Btk-overexpressing B cells showed no differences required for its activation (Rawlings et al., 1996). in Btk tyrosine phosphorylation after either BCR 1967 A.M.Scharenberg et al. Fig. 5. PtdIns-3,4,5-P interaction with the Btk PH domain induces Btk autophosphorylation. (A) Active p110 is able to induce Btk autophosphorylation. Top left and top right panels: NIH 3T3 fibroblasts were infected for 6 h with the indicated vaccinia viruses, harvested and resuspended at 10 cells/ml in calcium buffer. Cells were then quickly spun down and lysed, and post-nuclear supernatants were immunoprecipitated with the indicated antibodies or analyzed directly. Bottom left panel: NIH 3T3 cells were infected as indicated, and 100 nM wortmannin was added as indicated to the infection media for the last1hof infection. Cells were then processed in the same fashion as in the top panels. Bottom right panel: A20 B cells were infected for 14 h with the indicated vaccinia viruses and then harvested, stimulated and lysed as in Figure 4. Post-nuclear supernatants subsequently were immunoprecipitated with the indicated antibodies or analyzed directly. (B) p110-dependent Btk phosphorylation requires kinase activity and an intact PH domain but not an SH2 domain. Top panel: A20 B cells were infected for 14 h with the indicated vaccinia viruses and then otherwise analyzed as in the bottom right panel of (A). Bottom panel: NIH 3T3 cells were infected for 6 h with the indicated viruses, and then otherwise analyzed as in the top left panel of (A). engagement alone or BCR–FcγRIIb1 co-engagement examined the effect of SHIP expression on both Lyn- (data not shown). We therefore reasoned that perhaps induced and Lyn–p110-induced tyrosine phosphorylation only a small fraction of the total Tec kinases in the of Btk in NIH 3T3 cells. Co-expression of SHIP, but cells was participating in the receptor-induced signaling not a catalytically inactive SHIP mutant, was able events. In order to produce an environment in which to block Lyn-induced Btk tyrosine phosphorylation the high local concentration of signaling proteins likely (Figure 6D, left), and Lyn–p110-induced Btk phospho- to be present in BCR–FcγRIIb1 complexes would be rylation (Figure 6D, right top). The expression of SHIP able to affect the majority of the expressed Btk, we was also associated with a complete loss of the p110- 1968 PIP /Tec kinase calcium signaling pathway dependent PtdIns-3,4,5-P signal, confirming the primary activated by an upstream kinase. In the case of Akt, role of PtdIns-3,4,5-P among other D3 phosphoinositides PtdIns-3,4-P interacts with the Akt PH domain and 3 2 in Tec kinase activation (Figure 6D, right bottom). induces its partial activation. This mechanism is enhanced further by a second serine/threonine kinase, PDK-1, which is able to phosphorylate Akt and lock it into a fully active Discussion state. In the case of Btk, the prototype Tec kinase, We have presented genetic, pharmacological and biochem- interaction of its PH domain with PtdIns-3,4,5-P is ical evidence that PtdIns-3,4,5-P initiates Tec kinase sufficient to produce Btk autophosphorylation in a cellular activation and subsequent PLCγ2 tyrosine phosphorylation context. This effect is enhanced further by Src family and IP production. Our results provide a mechanistic kinases, at least in part through their ability to phosphoryl- link between D3 phosphoinositides and regulation of ate Btk within its activation loop (Rawlings et al., 1996). intracellular calcium levels, and represent only the second Therefore, while interacting with distinct effector proteins example of a cellular process which has been shown to in the context of quite different cellular processes, PtdIns- be controlled directly through an interaction with a D3 3,4-P and PtdIns-3,4,5-P appear to utilize functionally 2 3 phosphoinositide, the other being the activation of the Akt equivalent mechanisms in which they promote the mem- serine/threonine kinase during its role in anti-apoptotic brane association of their respective target kinase for the signaling (reviewed in Toker and Cantley, 1997). The purpose of promoting its activation by a second kinase. mechanisms through which Akt and Tec kinases are Taking into account what has been shown previously activated by interaction with D3 phosphoinositides appear regarding the mechanism of Btk activation, a complete to be strikingly analogous: in each case, phosphoinositide molecular mechanism for a Tec kinase calcium signaling binding to the PH domain mediates their ability to be pathway emerges based on our data: PtdIns-3,4,5-P initi- ates Tec kinase activation in concert with Src kinases, as well as probably targeting the kinase to the plasma membrane. Once the Tec kinase is bound to PtdIns-3,4,5- P and activated, an interaction between its SH2 domain and a tyrosine-phosphorylated ligand induced by BCR engagement is required to co-localize the activated Tec kinase and PLCγ2 for the purpose of tyrosine phosphorylat- ing PLCγ2. The importance of the latter interaction is supported by (i) the normal to enhanced PtdIns-3,4,5-P - induced activation of the Btk-R307K SH2 mutant coupled with its inability to participate in BCR-mediated PLCγ2 tyrosine phosphorylation; and (ii) our previous results indicating that the Btk SH2 domain is required for Btk (activated by co-expression with Lyn) to phosphorylate Fig. 6. SHIP-dependent PtdIns-3,4,5-P degradation blocks PtdIns- 3,4,5-P /Tec kinase-dependent signaling. (A) Co-engagement of the BCR and FcγRIIb1 blocks BCR-induced PtdIns-3,4,5-P accumulation. Uninfected A20 B cells were labeled with P and prepared for stimulation as above, then stimulated with either 15 μg/ml of Fab rabbit anti-mouse IgG or 30 μg/ml of intact rabbit anti-mouse IgG, followed by extraction and analysis of lipids as in Figure 3 above. Similar results were obtained with infected cells, although the magnitude of the Fab -stimulated PtdIns-3,4,5-P production was 2 3 reduced. (B) Co-engagement of the BCR and FcγRIIb1 blocks Tec kinase-dependent calcium signaling. A20 B cells were infected either with control virus or Btk, loaded with fura-2, and prepared for calcium assays as described above. Cells were then stimulated with either 15 μg/ml Fab or 30 μg/ml rabbit anti-mouse IgG, while fura-2 fluorescence was monitored by bulk spectrofluorimetry. (C) Co- engagement of the BCR and FcγRIIb1 blocks Tec kinase-dependent phosphorylation of PLCγ2. A20 B cells were infected either with control virus or Btk, stimulated with either 15 μg/ml Fab or 30 μg/ ml intact rabbit anti-mouse IgG, and PLCγ2 tyrosine phosphorylation was analyzed as described above. Identical results were obtained when stimulations were performed with intact rat anti-mouse IgG with or without pre-treatment with 2.4G2 (to block binding of the Fc fragment of intact rat IgG with the inhibitory Fc receptors), demonstrating that the differences in observed PLCγ2 tyrosine phosphorylation are not due to differences in the activation of the BCR. (D) SHIP blocks Lyn- and Lyn–p110-dependent activation of Btk. Left panel: Btk was expressed alone or with the indicated viruses, followed by analysis of phosphotyrosine content as in Figure 5. Right panel: Lyn, Btk and p110 were expressed with SHIP or with a catalytically inactive form of SHIP in NIH 3T3 cells. Each sample was divided into two parts. One part was used for analysis of Btk phosphotyrosine content as in Figure 5 (top), while the second part was used for lipid extraction and analysis of the level of PtdIns-3,4,5-P as in (A) (bottom). 1969 A.M.Scharenberg et al. PLCγ2 when they are co-expressed in fibroblasts in the the effect of FcγRIIb1 was first described. While FcγRIIb1 absence of receptor stimulation (Fluckiger et al., 1998). is known to recruit SHIP, it has been unclear whether SHIP- In the case of BCR-mediated signals, candidate Tec SH2 mediated breakdown of IP (which has been linked to cal- ligands would be the Syk tyrosine kinase, which is also cium influx in some cell types) or PtdIns-3,4,5-P is respons- required for BCR-mediated calcium mobilization, and ible for the block in sustained calcium signaling. The results which is capable of binding both PLCγ2 and Btk in vitro presented here demonstrate that engagement of FcγRIIb1 is (Takata et al., 1994; Law et al., 1996; Wan et al., 1997), associated with a lack of accumulation of PtdIns-3,4,5-P , or PLCγ2 itself. In other systems, other receptor-associated inhibition of Tec kinase-dependent PLCγ2 tyrosine kinases or a structural feature of the receptor itself might phosphorylation and inhibition of Tec kinase-dependent cal- provide the co-localizing signal. This mechanism provides cium signaling, and that SHIP is able to degrade PtdIns- a full molecular framework with which to predict how 3,4,5-P and block Tec kinase activation in a heterologous mutations in Btk which cause X-linked agammaglobuline- system. Together, these data suggest that SHIP-dependent mia are able to interrupt signaling: PH mutations block Btk degradation of PtdIns-3,4,5-P in the local neighborhood membrane targeting and all subsequent events including its of BCR signaling complexes blocks calcium signaling by activation, SH2 mutations block the ability of membrane producing local deactivation of Tec kinases and consequent targeted/activated Btk to co-localize with PLCγ2 or other decreased PLCγ2 activation, as well as loss of the cofactor/ substrates, and kinase domain mutations block the ability substrate access functions of PtdIns-3,4,5-P (as discussed of membrane targeted/co-localized Btk to tyrosine phos- above). As demonstrated in Fluckiger et al. (1998), the phorylate PLCγ2 or other substrates. resulting inhibition of IP production allows an initial endo- The ability of PtdIns-3,4,5-P to initiate Tec-dependent plasmic reticulum (ER) calcium release followed by ER PLCγ2 tyrosine phosphorylation provides at least a partial calcium store refilling and so a loss of the store-operated molecular explanation for previous observations that wort- calcium influx required for sustained calcium signals, mannin is able to inhibit BCR-mediated IP production and accounting for the observed apparent selectivity of FcγRIIb1 calcium mobilization (Hippen et al., 1997; Kiener et al., for blocking calcium influx. 1997). In addition, our data further suggest that D3 phospho- In summary, our data implicate PtdIns-3,4,5-P as a inositides may play a second role in the regulation of PLCγ2, critical regulator of calcium signaling at least in part either alone or with Tec kinases. Comparison of the effect through its ability to initiate Tec kinase activation and of wortmannin on PLCγ2 tyrosine phosphorylation resulting tyrosine phosphorylation of PLCγ2 and IP (Figure 5C, compare lanes 2 and 8) and calcium fluxes production. A role for PtdIns-3,4,5-P –PH domain inter- (Figure 2A and B) in cells expressing Btk alone but not actions in Tec kinase-dependent PLCγ activation provides treated with wortmannin with those in cells co-expressing a molecular basis for why mutations which eliminate this p110–Btk but treated with wortmannin, shows similar levels interaction in the B cell-specific Tec member Btk cause of PLCγ2 tyrosine phosphorylation but markedly different aberrant B-cell development in humans and mice, and a sustained levels of intracellular calcium. This implies that molecular explanation for the inhibition of calcium in addition to tyrosine phosphorylation, PLCγ2 requires signaling mediated by the association of the SHIP inositol another factor(s) to be present for it to hydrolyze its PtdIns- 5-phosphatase with the inhibitory receptor FcγRIIb1. As 4,5-P substrate to IP effectively, a finding consistent with exemplified by BCR–FcγRIIb1 co-engagement, the PtdIns- 2 3 the normal levels of BCR-induced PLCγ2 tyrosine phospho- 3,4,5-P /Tec kinase calcium signaling pathway provides a rylation found in XLA-derived B-cell lines (Fluckiger et al., mechanism for cells to control the magnitude of calcium 1998). Assuming that wortmannin is only blocking D3 phos- signals initiated by receptors which utilize PLCγ isozymes phoinositide production, this second factor may involve independently of the strength of the external stimulus to direct regulation of PLCγ1/2 by PtdIns-3,4,5-P , as has been those receptors, and may explain the apparent involvement suggested for PLCγ1 (Rhee and Bae, 1997; Falasca et al., of PI3K/Tec signaling in signaling through co-stimulatory 1998). However, we favor the possibility that the second receptors such as CD19 or CD28. The tissue distribution factor involves PtdIns-3,4,5-P acting together with Tec of Tec kinases, the diversity of receptors capable of kinases independently of their activity based on the follow- activating them, and the recent appreciation of the signi- ing: (i) the relatively small effect of overexpression of p110 ficance of receptor-mediated inhibitory signals involving alone on IP and calcium (see Figure 1); (ii) the lack of inositol 5-phosphatases (Scharenberg and Kinet, 1996) dominant-negative effect on IP and calcium when inactive suggest that the PtdIns-3,4,5-P /Tec signaling mechanism 3 3 Btk is overexpressed (see Figure 2); and (iii) a Btk-deficient is likely to be of broad importance. form of the DT-40 cell line which produces no calcium signal, but regains a partial signal when it is re-transfected Materials and methods with inactive Btk (Takata and Kurosaki, 1996). A potential unifying explanation for these data is that PtdIns-3,4,5-P Cell culture, transformation assays, recombinant virus production and cDNAs functions as an inducible ‘tag’ for caveolae islands of A20 B cells were grown in RPMI-1640 with 10% fetal bovine serum PtdIns-4,5-P (Pike and Casey, 1996); and that PtdIns-3,4,5- 2 –5 and 10 M 2-mercaptoethanol. NIH 3T3 cells were grown in Dulbecco’s P /Tec kinase complexes function as adaptors to bring modified Eagle’s medium (DMEM) with 10% calf serum. PtdIns-4,5-P into proximity with activated PLCγ2. Identi- A20 infections were performed by adding 5 p.f.u./cell of recombinant virus to ~20% confluent A20 cells and allowing infections to proceed fying which of the above scenarios best explains PLCγ1/2 for 12–15 h. Where appropriate, control recombinant virus was added function is an important goal for future investigations. so that all samples were exposed to an equal number of p.f.u./cell. A The mechanism by which engagement of FcγRIIb1 with recombinant virus containing a cDNA encoding human Gβ1 inserted in the BCR or other antigen receptor is able to inhibit sustained an antisense orientation was used as the control virus because the calcium signals has been the subject of much interest since transcript generated was similar in length to that of Btk. 1970 PIP /Tec kinase calcium signaling pathway The cDNAs for p110α, Tec, wild-type Btk and all mutant forms of functions of IgG Fc receptors in B lymphocytes. Science, 256, Btk have been described previously (Hiles et al., 1992; Rawlings et al., 1808–1812. 1996; Fluckiger et al., 1998). The cDNA for the p110* construct has Andreotti,A.H., Bunnell,S.C., Feng,S., Berg,L.J. and Schreiber,S.L. also been described previously (Hu et al., 1995). (1997) Regulatory intramolecular association in a tyrosine kinase of The retrovirus expressing p110* was constructed by subcloning the the Tec family. Nature, 385, 93–97. p110* cDNA into the pSRαMSVtk-neo retroviral recombination vector Auger,K.R., Serunian,L.A., Soltoff,S.P., Libby,P. and Cantley,L.C. (1989) and selecting for recombinant retroviruses. Procedures used for the PDGF-dependent tyrosine phosphorylation stimulates production of production of retroviruses for transformation assays have been described, novel polyphosphoinositides in intact cells. Cell, 57, 167–175. as has the recombinant Btk retrovirus and the procedures used for the Bijsterbosch,M.K. and Klaus,G.G.B. (1985) Crosslinking of surface transformation assays (Afar et al., 1996). immunoglobulin and Fc receptors on B lymphocytes inhibits The p110-expressing recombinant vaccinia virus was constructed by stimulation of inositol phospholipid breakdown via the antigen subcloning a p110α cDNA into the pSC-66 vaccinia recombination receptors. J. Exp. Med., 162, 1825–1836. plasmid. 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(1996) Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton’s tyrosine kinase. EMBO J., 15, 6241–6250. Scharenberg,A.M. and Kinet,J.-P. (1996) The emerging field of receptor- mediated inhibitory signaling: SHP or SHIP? Cell, 87, 961–964. Scharenberg,A.M., Lin,S., Cuenod,B., Yamamura,H. and Kinet,J.-P. (1995) Reconstitution of interactions between tyrosine kinases and http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3)/Tec kinase‐dependent calcium signaling pathway: a target for SHIP‐mediated inhibitory signals

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Springer Journals
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Copyright © European Molecular Biology Organization 1998
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0261-4189
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1460-2075
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10.1093/emboj/17.7.1961
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Abstract

The EMBO Journal vol.17 no.7 pp.1961–1972, 1998 Phosphatidylinositol-3,4,5-trisphosphate (PtdIns- 3,4,5-P )/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals with the ability to interact with PtdIns-3,4,5-P or PtdIns- Andrew M.Scharenberg, Ousama El-Hillal, 3,4-P in vitro have now been described (Rameh et al., David A.Fruman , Laurie O.Beitz, 2 2 3,4 1995, 1997; Klarlund et al., 1997). Recently, the task of Zuomei Li , Siqi Lin, Ivan Gout , 1 5 determining whether and/or how each of these in vitro Lewis C.Cantley , David J.Rawlings and interactions translate into cellular functions was begun by Jean-Pierre Kinet three different groups who provided evidence that PtdIns- Laboratory of Allergy and Immunology and Laboratory of Signal 3,4-P and PtdIns-3,4,5-P act as upstream activation 2 3 Transduction, Beth Israel Deaconess Medical Center and Harvard signals for the AKT serine/threonine kinase via an inter- Medical School, Boston, MA 02215, Department of Microbiology action with its pleckstrin homology (PH) domain (Franke and Molecular Genetics, University of California at Los Angeles, 5 et al., 1995, 1997; Alessi et al., 1997; Klippel et al., 1997; Los Angeles, CA 90095-1662, Department of Pediatrics, University Stokoe et al., 1997). The cellular functions attributable to of California at Los Angeles, Los Angeles, CA 90095-1752, USA, Ludwig Institute for Cancer Research, 91 Riding House Street, other in vitro protein–PtdIns-3,4,5-P /PtdIns-3,4-P inter- 3 2 London W1P 8BT, UK and Institute of Molecular Biology and actions remain uncharacterized, but the diversity of pro- Genetics, Kyiv, Ukraine teins which carry these domains suggests that PtdIns- Corresponding author 3,4,5-P /PtdIns-3,4-P are likely to act in an analogously 3 2 e-mail: [email protected] diverse range of processes depending on the cellular context in which they are produced. Tec family non-receptor tyrosine kinases have been The PH domain of Bruton’s tyrosine kinase (Btk), a implicated in signal transduction events initiated by member of the Tec non-receptor tyrosine kinase family, cell surface receptors from a broad range of cell types, is among the protein domains capable of interacting with including an essential role in B-cell development. A PtdIns-3,4,5-P in vitro (Salim et al., 1996; Rameh et al., unique feature of several Tec members among known 1997). Tec kinases are an emerging family of proteins tyrosine kinases is the presence of an N-terminal which are expressed in both hematopoietic and non- pleckstrin homology (PH) domain. We directly demon- hematopoietic tissues. Four Tec members (Tec, Btk, Itk strate that phosphatidylinositol-3,4,5-trisphosphate and Bmx) have closely homologous structures which (PtdIns-3,4,5-P ) interacting with the PH domain acts include an N-terminal PH domain, followed by SH3, SH2 as an upstream activation signal for Tec kinases, and tyrosine kinase domains (reviewed in Desiderio and resulting in Tec kinase-dependent phospholipase Cγ Siliciano, 1994; Rawlings and Witte, 1995). These Tec (PLCγ) tyrosine phosphorylation and inositol tris- kinases have been implicated in the signaling pathways phosphate production. In addition, we show that this of a variety of hematopoietic receptors, including several pathway is blocked when an SH2-containing inositol types of cytokine and antigen receptors (reviewed in phosphatase (SHIP)-dependent inhibitory receptor is Rawlings and Witte, 1995). The mechanism of their engaged. Together, our results suggest a general mech- activation by these receptors is thought to involve a two- anism whereby PtdIns-3,4,5-P regulates receptor- step process in which they receive a currently undefined dependent calcium signals through the function of signal which targets them to the vicinity of activated Src Tec kinases. kinases, after which their activation occurs through Keywords: B-cells/inositol trisphosphate/phospholipase a transphosphorylation/autophosphorylation mechanism C/receptor/tyrosine kinases (Mahajan et al., 1995; Rawlings et al., 1996). In terms of downstream targets, Btk is thought to play an important role in apoptotic signaling as well as the activation of Jnk kinases and phospholipase Cγ2 (PLCγ2) (Rigley et al., Introduction 1989; Takata and Kurosaki, 1996; Uckun et al., 1996; Kawakami et al., 1997). Furthermore, B-cell overexpres- Receptors of virtually every type have been shown to sion experiments show that participation in PLCγ2 activa- stimulate the production of the phosphoinositides phos- tion is a general property of Tec kinases, that their role phatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P ) and in PLCγ2 activation is distinct from that of Src or Syk PtdIns-3,4-P (Auger et al., 1989; Serunian et al., 1991; family kinases, and that they are particularly important Gold and Aebersold, 1994; Gold et al., 1994; Okada et al., for producing the sustained production of inositol tris- 1996). These lipids are produced by the action of a family phosphate (IP ) required for store-operated calcium influx of enzymes which specifically phosphorylate the D3 (Fluckiger et al., 1998). position of the inositol ring in phosphoinositides (reviewed While progress has been made in identifying signaling in Carpenter and Cantley, 1996; Vanhaesebroeck et al., 1997), and are thought to act as inducible membrane pathways lying upstream and downstream from Tec kin- targeting signals for proteins which carry domains capable ases, little is known about Tec kinase structure–function of interacting with them. Several examples of domains relationships. Many investigators have attempted to under- © Oxford University Press 1961 A.M.Scharenberg et al. stand Tec signaling mechanisms by identifying ligands for for their ability to transform rat fibroblasts synergistically the subdomains of these kinases. Four potential cellular with Btk. This screen involves assaying the transforming ligands for Tec family PH domains have emerged from activity of Btk alone or in combination with other molec- in vitro studies including: (i) G-protein β/γ subunits ules using retroviral expression of the proteins of interest (Tsukada et al., 1994); (ii) protein kinase C (most isoforms) in a soft agar growth transformation assay, and has (Yao et al., 1994; Kawakami et al., 1995); (iii) BAP-135, previously permitted receptor-independent analysis of Btk a 135 kDa protein of unknown function (Yang and activation and signaling (Afar et al., 1996). Using this Desiderio, 1997); and (iv) inositol-1,3,4,5-tetrakisphos- assay, Btk or an activated form of p110, known as p110* phate (IP , a soluble inositol phosphate) and PtdIns-3,4,5- (Hu et al., 1995), each produce 10 colonies per plate, P (Fukuda et al., 1996; Salim et al., 1996; Rameh et al., while their co-expression results in an ~6-fold enhance- 1997). Several proteins, including CBL, WASP and SAM- ment in number of colonies. In contrast, co-expression of 68 among others, have been proposed to function in Tec Btk with Cbl or Vav, both of which have also been shown family signaling via their ability to interact with Tec to interact with Btk in vitro, does not produce a similar family SH3 domains in vitro (Cory et al., 1995; Bunnell enhancement (Z.Li, D.J.Rawlings, H.Park and O.N.Witte, et al., 1996; Andreotti et al., 1997). Finally, Tec has been unpublished data). proposed to function in linking cytokine receptors to Vav (through a direct association) and phosphatidylinositol 3- p110–Tec kinase co-expression synergistically kinase (PI3K)-dependent signaling pathways (through an enhances BCR-mediated IP production and association with the p85 subunit of PI3K) based on co- calcium signals precipitation and in vitro binding data (Machide et al., Increased expression of Tec kinases in B cells produces 1995; Takahashi-Tezuka et al., 1997). It is currently an enhanced calcium signal characterized in particular by unclear whether and/or how each of the binding inter- sustained calcium influx (Fluckiger et al., 1998). Based actions described for various Tec family subdomains are on the results of the transformation screen, we investigated integrated into the signaling function of these kinases. whether the p110 subunit of PI3K could functionally However, compelling evidence for the relevance of PH interact with Tec kinases during calcium signaling domain function to Tec kinase function exists in the form (Figure 1A). In a typical experiment, p110 expression of spontaneously arising mutations in the Btk PH domain produces only a minor change in the overall calcium flux which cause X-linked agammaglobulinemia in humans relative to control infections, while Btk, Itk and Tec and mice (reviewed in Conley and Rohrer, 1995; Rawlings expression produce an enhanced calcium signal most and Witte, 1995). Furthermore, one such mutation, an evident at later time points. However, when expressed arginine to cysteine substitution at position 28, has been together with p110, the effects of Btk, Itk and Tec shown to reduce the in vitro inositol polyphosphate- and are substantially larger and more sustained—reproducibly phosphoinositide-binding activity of the isolated Btk PH greater than the additive effects of p110 and the kinase domain (Fukuda et al., 1996; Salim et al., 1996; Rameh alone. Blots of the lysate (data not shown) demonstrate et al., 1997). that similar amounts of each kinase are expressed when Given the confusing picture of Tec kinase function Btk, Itk and Tec are expressed alone or with p110. which has emerged from studying the interactions of its In order to test whether the PI3K activity of p110 and various domains in vitro, we undertook a functional not some other factor related to its increased expression approach to studying Tec kinase signaling by examining level was responsible for the observed synergistic effects how co-expressing them with other signaling proteins on the calcium signal, we examined the effect of wortman- affects Tec kinase-regulated signaling events in vivo. This nin on the calcium flux generated by expression of Tec approach identified an activated form of the p110 subunit kinases alone or in combination with p110 (Figure 1B, of PI3K (p110) as a prominent synergizing molecule when note that the traces in this experiment were obtained from expressed with the Tec kinase prototype Btk in a fibroblast the same experiment as the Btk traces without wortmannin transformation assay. Subsequent expression of Btk, Itk pre-treatment in Figure 1A). As expected and as previously and Tec with p110 in B cells showed a similar functional reported, wortmannin inhibits the calcium fluxes of unin- synergy at the level of the B-cell receptor (BCR)-initiated fected A20 B cells and of A20 B cells infected with a calcium signal, and this system was used for a detailed control recombinant vaccinia virus (Kiener et al., 1997; molecular analysis of the synergy mechanism. The results A.M.Scharenberg and J.-P.Kinet, unpublished data; of this analysis suggest that PtdIns-3,4,5-P initiates Figure 1B). Wortmannin is also able to reverse the minor PLCγ2-dependent IP production at least in part through enhancement of calcium flux seen with infection with the its ability to interact with and activate Tec kinases. In p110 virus alone, indicating that the effect of p110 on addition, we show that FcγRIIb1, an inhibitory receptor calcium mobilization is mediated through its phosphoinosi- which recruits the SHIP inositol 5-phosphatase (Ono tide metabolites. In addition, wortmannin inhibits essen- et al., 1996), eliminates BCR-induced PtdIns-3,4,5-P tially all of the calcium flux enhancement generated by accumulation and results in blocked Tec kinase-dependent Btk expression alone, and substantially but incompletely calcium signaling. inhibits the effect of p110–Btk co-expression. The com- plete inhibition of the effect of Btk expressed alone has Results the important implication that the action of endogenous p110-PI3K synergizes with Btk in a transformation PI3K is required for Btk to function in calcium signaling. assay The less complete inhibition of calcium mobilization in In order to identify signaling proteins which functionally Btk–p110-expressing cells is in part expected, as the interact with Tec kinases, proteins of interest were screened amount of wortmannin used would produce less complete 1962 PIP /Tec kinase calcium signaling pathway Fig. 1. p110–Tec kinase co-expression synergistically enhances BCR-mediated IP production and calcium signals. (A) p110–Tec kinase co- expression produces synergistically increased calcium signals. Upper panels: A20 B cells were plated to 20% confluency and subsequently infected for 13 h with the indicated combinations of recombinant vaccinia viruses. The following morning, the cells were harvested, loaded with fura-2, and calcium mobilization in response to 15 μg/ml Fab rabbit anti-mouse IgG was measured in a bulk spectrofluorimeter at 30°C. (B) Btk-dependent enhancement of calcium signaling is inhibitable by wortmannin. The same experiment as (A), except that cells were pre-treated with 100 nM wortmannin for 10 min prior to the assay. (C) p110–Btk co-expression produces a synergistically increased IP signal. Cells were infected and harvested identically to those in (A). After harvest, they were suspended at 510 cells/ml in calcium buffer and stimulated with 30 μg/ml of Fab rabbit anti-mouse IgG. After the indicated times of stimulation, cells were spun down rapidly and lysed in 20% trichloroacetic acid (w/v in H O), and prepared for IP radioreceptor assay as specified in the manufacturer’s protocol. inactivation of PI3K activity in these cells because its examined whether p110 would be able to complement the pharmacologic target, the p110 PI3K subunit, is present signaling capabilities of mutant versions of Tec kinases. in greater abundance. Similar results are obtained with Because of the availability of a variety of Btk mutants in other Tec kinases in the A20 cells (data not shown). our laboratory, Btk was again used as the prototype Tec Tec kinase-dependent enhancement of calcium signaling kinase for these experiments. Btk constructs carrying occurs at least in part via enhanced production of IP inactivating mutations within each major domain [kinase (Fluckiger et al., 1998). Using Btk as a prototype, we (K430R), PH-R28C, ΔSH3 (full deletion) and SH2-R307K evaluated whether increased Tec kinase expression in (Figure 2A–D)] were expressed in A20 B cells, and combination with p110 was affecting IP production calcium flux (left panel) and IP generation (right panel) 3 3 (Figure 1C). Control infected A20 cells produce an ~2- were analyzed for expression of each mutant kinase alone fold increase in IP at 1 min after BCR engagement, or with p110 (designated black traces). Traces representing which then decays back to just above baseline by 10 min. control and p110 alone infections for each experiment are While expression of p110 alone increases IP production shown in gray for purposes of comparison. As shown by only slightly relative to control, Btk expression alone the designated black traces in Figure 2A, the catalytically produces a substantial enhancement, and p110–Btk co- inactive mutant expressed alone is not able to enhance expression results in synergistically higher and more the calcium (left panel) or IP signals (right panel) sustained IP production than Btk alone. Therefore, the significantly, and co-expression with p110 is not able to synergy between p110 and Tec kinases observed at the complement its loss of signaling function. Of the Btk level of the calcium signal correlates well with the interaction domain mutants, the Btk-ΔSH3 produces both production of IP , suggesting that p110 and Tec kinases enhancement of signaling when expressed alone and are both somehow involved in the regulation of PLCγ2, synergistic signaling when co-expressed with p110 the principal PLC isozyme utilized during BCR signaling. (Figure 2C, direct comparison of the ΔSH3 and wild-type Btk in other experiments showed that the ΔSH3 mutant p110–Tec synergy requires intact PH, SH2 and reproducibly generated similar but somewhat larger effects kinase domains than wild-type Btk, data not shown). The R28C (Figure 2B) In order to begin to analyze the mechanism through which and R307K (Figure 2D) mutants were unable to enhance p110 and Tec kinases were acting on IP production, we calcium signaling nor did they show significant comple- 1963 A.M.Scharenberg et al. effects of activation of other kinases, the direct products of p110-PI3K action are known—PtdIns-3,4,5-P and PtdIns-3,4-P . Therefore we decided to begin determining which of the above scenarios best explained the p110– Tec synergy by testing the hypothesis that Tec kinases were enhancing p110 signaling. One implication of the hypothesis that Tec kinases act upstream from p110 would be that production of the direct p110 product would be increased. Available evidence suggests that PtdIns-3,4,5-P is the primary direct product generated by p110 (via 3 phosphorylation of PtdIns-4,5- P ) while PtdIns-3,4-P is generated indirectly via 5 2 2 dephosphorylation of PtdIns-3,4,5-P (Stephens et al., 1991). Therefore, we first examined the effect of co- expression of p110 and Btk on the basal and stimulated levels of PtdIns-3,4,5-P in A20 B cells (Figure 3A). In control cells, PtdIns-3,4,5-P is just detectable in our TLC assay after 2 min of stimulation, consistent with what has been previously published on PtdIns-3,4,5-P production in tumor B-cell lines (Gold and Aebersold, 1994). This production is enhanced significantly by expression of p110 alone, and increased marginally by expression of Btk alone at these expression levels (at higher expression levels achieved by longer infections, significant elevations in PtdIns-3,4,5-P are detectable with Btk expression alone; A.M.Scharenberg, O.El-Hillal and J.-P.Kinet, unpublished observations; see also Figure 3C below). However, co- expression of Btk and p110 results in much higher levels of PtdIns-3,4,5-P than p110 alone or Btk alone in both basal and stimulated conditions, indicating the presence of a p110–Tec kinase-dependent synergy on the level of cellular PI3K lipid products. As substitution of other Tec kinases in these experiments produced identical synergy effects (data not shown), we focused on Btk for subsequent experiments because of the ready availability of Btk Fig. 2. p110–Tec synergy requires intact Btk PH, SH2 and kinase mutants and immunological reagents in our laboratory. domains. (A–D) For calcium assays, A20 B cells were infected with A second implication of the hypothesis that Tec kinases the indicated vaccinia viruses, harvested, loaded with fura-2, act upstream from p110 is that loss-of-function Tec mutants stimulated and assayed in a manner identical to those in Figure 1. For would be expected to show a loss of the synergy at the IP assays, A20 B cells were infected with the indicated vaccinia viruses, harvested, stimulated, lysed in 20% TCA, and processed for level of PtdIns-3,4,5-P . Therefore, we compared the IP assay in a manner identical to those in Figure 1. Note that for both PtdIns-3,4,5-P signal generated by co-expression of p110 the calcium and IP assays, the K430R, R28C and R307K mutants and wild-type Btk with that generated by co-expression were assayed in the same experiment, so that their control and p110 of p110 and inactive Btk (Btk-K430R, which is unable to alone curves (shown in gray) are identical. The ΔSH3 was assayed in calcium signal and is not complemented by p110, see a separate experiment, so the control curves from that particular experiment are shown for comparison. Figure 2) (Figure 3B, left panel). Similar experiments were performed in NIH 3T3 fibroblasts in the absence of mentation of their signaling defects when expressed with receptor stimulation (right panel). These experiments show p110. that the inactive Btk mutant has equivalent or greater ability than wild-type Btk to enhance the PtdIns-3,4,5-P p110–Tec co-expression synergistically enhances signal. They also demonstrate that no hematopoietic- detectable PtdIns-3,4,5-P specific component is required for the synergistic PtdIns- Based on published reports of ligands of the various 3,4,5-P signal. domains of Tec kinases (see Introduction) and the recent Again assuming that Tec kinases act upstream from suggestion that p110 might be a cofactor for PLCγ p110, the ability of inactive Btk to produce synergistic activation (Rhee and Bae, 1997), the p110–Tec kinase accumulation of PtdIns-3,4,5-P when co-expressed with synergy could reasonably be explained by four possible p110 could only be accounted for if p110 activation was mechanisms: (i) p110-dependent enhancement of Tec being enhanced by Btk in a manner which was independent kinase signaling; (ii) Tec kinase-dependent enhancement of Btk activity. In order to address this possibility, we used of p110 signaling; (iii) a combination of both of these HPLC to analyze the effect of increased Btk expression on mechanisms; or (iv) a cooperative mechanism in which both PtdIns-3,4,5-P and PtdIns-3,4-P (Figure 3C, left 3 2 Tec kinases and p110 act independently on PLCγ2 function. and right panels). Increased Btk expression is associated While identifying direct targets of protein kinases can be with a large (~3-fold) increase in PtdIns-3,4,5-P levels difficult in a cellular context due to the confounding with essentially no effect on PtdIns-3,4-P levels. Since 1964 PIP /Tec kinase calcium signaling pathway Fig. 3. p110–Tec co-expression synergistically enhances detectable PtdIns-3,4,5-P .(A) p110–Btk co-expression produces a synergistic enhancement of the amount of detectable PtdIns-3,4,5-P (PIP3). Both panels: A20 B cells were infected for 13 h with the indicated vaccinia viruses, harvested, labeled with P and stimulated or not for 2 min with 30 μg/ml Fab rabbit anti-mouse IgG. Lipids were then extracted and analyzed by TLC. (B) Btk kinase activity is not required to produce an enhanced PtdIns-3,4,5-P signal. Left panel: A20 B cells were infected with the indicated vaccinia viruses, and then analyzed for PtdIns-3,4,5-P levels as in (A). Right panel: NIH 3T3 fibroblasts were infected for 6 h with the indicated vaccinia viruses, harvested and labeled with P. Lipids were then extracted and analyzed by TLC. (C) Increased Btk expression does not significantly affect production of PtdIns-3,4-P . A20 B cells were infected for 16 h with Btk alone or control vaccinia viruses. Cells were then harvested, labeled with P, stimulated or not for 2 min with 30 μglml Fab rabbit anti-mouse IgG and lipids were extracted and analyzed by HPLC. Note that expression when using a single virus is typically greater than when co-infections are performed, so that Btk expression in these experiments was in the range of 2-fold greater than in all other experiments. (D) An intact PH domain but not SH2 domain is required to produce synergistic enhancement of PtdIns-3,4,5-P . Left panel: A20 B cells were infected for 13 h with the indicated vaccinia viruses, and then analyzed for PtdIns-3,4,5-P levels as in (A). Right panel: NIH 3T3 fibroblasts were infected for 6 h with the indicated vaccinia viruses, and then otherwise processed as in (B). p110 activation typically produces proportional accumula- The remaining explanation for the ability of increased Tec tion of both PtdIns-3,4,5-P and PtdIns-3,4-P (Hu et al., kinase expression to increase the accumulation of PtdIns- 3 2 1995; Klippel et al., 1996), the selective enhancement in 3,4,5-P was that it was somehow reducing the rate of PtdIns-3,4,5-P levels suggests that Btk is not significantly PtdIns-3,4,5-P degradation. Since Btk is known to be 3 3 affecting cellular PI3K activity. capable of binding PtdIns-3,4,5-P via its PH domain, Based on the above two experiments, we felt that it the simplest explanation for how increased Tec kinase was unlikely that Tec kinases were acting upstream to expression could decrease PtdIns-3,4,5-P degradation was activate p110 and enhance D3 phosphoinositide synthesis. by binding to PtdIns-3,4,5-P and thereby protecting it 1965 A.M.Scharenberg et al. from endogenous inositol phosphatases. We therefore tested which interaction domains of Btk were participating in the enhancement of PtdIns-3,4,5-P accumulation by expressing the R28C and R307K Btk mutants alone or with p110-PI3K in A20 B cells (Figure 3D, left panel). The R28C PH domain mutation (which greatly reduces the binding of the isolated Btk PH domain to PtdIns- 3,4,5-P in vitro) eliminates the enhancement of the PtdIns-3,4,5-P signal seen after co-expression of p110. In contrast, an enhanced PtdIns-3,4,5-P signal identical to that of wild-type Btk is seen for the R307K SH2 FLVR mutant. A comparable result is obtained if the co- expression is performed in fibroblasts (right panel), again confirming that additional hematopoietic-specific compon- ents are not required. This experiment also shows that the ability of the ΔSH3 mutant to enhance PtdIns-3,4,5-P detection is comparable with that of other forms of Btk (right panel), consistent with its containing an intact PH domain and its ability to produce enhanced calcium signaling. Finally, together with the lack of significant enhancement of PtdIns-3,4-P accumulation in the HPLC experiment in Figure 3B above, this experiment provides in vivo data to support the previously described binding specificity of the Btk PH domain for PtdIns-3,4,5-P . Btk-dependent PLCγ2 tyrosine phosphorylation is PI3K-dependent The above data demonstrate that Tec kinase PH domains are interacting with PtdIns-3,4,5-P in the context of our co-expression system, and that Tec kinases do not act as major upstream activation signals for p110. They therefore constrain the potential mechanisms for the functional synergy between Tec kinases and p110 to those in which PtdIns-3,4,5-P acts either upstream from or together with the Tec kinase to promote IP production and calcium mobilization through the activation of PLCγ2. Since Btk is known to participate in the tyrosine phosphorylation of PLCγ2, we first evaluated whether the synergistic effect on PLCγ2 activation (as assessed by IP Fig. 4. Btk-dependent PLCγ2 tyrosine phosphorylation is PI3K- production) could be accounted for via synergistic effects dependent. (A) BCR-mediated PLCγ2 tyrosine phosphorylation is on PLCγ2 tyrosine phosphorylation. Using conditions enhanced by p110–Btk co-expression. A20 B cells were infected for identical to those used in the calcium synergy and IP 13 h with the indicated vaccinia viruses, harvested, washed once with calcium buffer, resuspended at 10 cells/ml in calcium buffer and experiments, we expressed various combinations of p110 stimulated or not for 2 min with 30 μg/ml Fab rabbit anti-mouse and Btk in the A20 B-cell line, and then analyzed the IgG. Cells were then quickly spun down and lysed, and post-nuclear level of BCR-induced PLCγ2 tyrosine phosphorylation supernatants were immunoprecipitated with the indicated antibodies or (Figure 4A). Relative to control infection, expression of directly analyzed. (B) p110–Btk-dependent PLCγ2 tyrosine phosphorylation requires kinase activity and intact PH and SH2 p110 alone produces a slight enhancement of PLCγ2 domains. A20 B cells were infected for 13 h with the indicated tyrosine phosphorylation, consistent with the idea that vaccinia viruses, and otherwise processed identically to those in (A). p110 is able to functionally interact with endogenous Btk. (C) Wortmannin treatment of A20 B cells overexpressing Btk or In contrast, Btk expression alone produces an easily p110–Btk inhibits PLCγ2 tyrosine phosphorylation. A20 B cells were detectable enhancement, which is then enhanced further infected for 13 h with the indicated vaccinia viruses, and otherwise processed identically to those in (A), except that the indicated samples by co-expression with p110. were pre-treated for 10 min with 100 nM wortmannin. If enhanced PLCγ2 tyrosine phosphorylation is respons- ible for the functional synergy between p110 and Btk, then PLCγ2 tyrosine phosphorylation should be eliminated of p110 to complement them in calcium signaling and by Btk mutants which eliminate the synergy seen at the IP assays. levels of IP and calcium. We tested this by expressing As a further test of the hypothesis that modulation of p110 alone or with wild-type or loss-of-function Btk PLCγ2 tyrosine phosphorylation by p110–Btk accounted mutants and analyzing BCR-induced PLCγ2 tyrosine for the synergistic enhancement of calcium signaling, we phosphorylation as above (Figure 4B). All loss-of-function treated cells overexpressing Btk or p110–Btk with 100 nM Btk mutations greatly reduce the enhancement of PLCγ2 wortmannin for 10 min and analyzed PLCγ2 tyrosine tyrosine phosphorylation produced by co-expression of phosphorylation (Figure 4C). Consistent with the effect p110 and wild-type Btk, consistent with the inability of wortmannin on the calcium signal (see Figure 1 1966 PIP /Tec kinase calcium signaling pathway above), PLCγ2 tyrosine phosphorylation is inhibited by In order to analyze the mechanism of p110-induced Btk wortmannin pre-treatment in both Btk- and p110–Btk- autophosphorylation, we examined the effect of the various overexpressing cells. The greater residual level of PLCγ2 Btk interaction domain mutations on the ability of p110 tyrosine phosphorylation in p110–Btk-overexpressing cells to induce Btk autophosphorylation (Figure 5B) in A20 B- than in cells expressing Btk alone may partially explain cell and fibroblast environments. The R28C PH domain why wortmannin does not completely revert the effect mutant eliminates p110-dependent Btk tyrosine phospho- of p110–Btk co-expression on the calcium signal (see rylation in both environments. In comparison, the level of Figure 1B above). Finally, the ability of wortmannin to tyrosine phosphorylation of the ΔSH3 mutant is similar attenuate the enhancement of PLCγ2 tyrosine phosphoryla- to that of wild-type Btk, and that of the R307K mutant is tion produced by Btk expression alone has the important markedly enhanced. Together with the results from Figures implication that Btk-dependent tyrosine phosphorylation 1–4, these results suggest that PtdIns-3,4,5-P acts as an of PLCγ2 is normally a PI3K-dependent process. upstream activation signal for Btk through an interaction with the Btk PH domain, and that the SH3 and SH2 PtdIns-3,4,5-P interaction with the Btk PH domain domains are dispensable for this process. It is important induces Btk autophosphorylation to note that the ΔSH3 mutant is missing one phosphoryla- The synergistic effect of p110–Btk co-expression on tion site at residue 223, so its phosphorylation level would PLCγ2 tyrosine phosphorylation suggests that co-expres- normally be expected to be ~50% of wild-type Btk (Park sion of Btk and p110 is either directly affecting Btk et al., 1996). The enhanced autophosphorylation observed activation or is somehow co-localizing Btk and PLCγ2, for the R307K mutant was unexpected, but we speculate with either scenario resulting in enhanced Btk-dependent that since the Btk SH2 domain is required for Btk- tyrosine phosphorylation of PLCγ2. We and others have dependent phosphorylation of PLCγ2, this mutation might shown previously that Btk activation, which is identical somehow inhibit access of Btk to substrates, thereby to that seen after BCR stimulation, can be induced by co- enhancing its opportunities for autophosphorylation. expression of Btk with Src kinases, and that this occurs by enhanced autophosphorylation of Btk (Mahajan et al., The FcγRIIB1–SHIP inhibitory receptor complex 1995; Rawlings et al., 1996). Since enhanced Btk activa- blocks PtdIns-3,4,5-P /Tec kinase-dependent tion would explain the enhanced PLCγ2 tyrosine phospho- calcium signaling rylation observed with p110–Btk co-expression, our initial The inhibitory receptor FcγRIIb1 is expressed on B investigations focused on whether p110 could be involved cells, where its co-engagement with the BCR results in in promoting Src kinase-induced Btk autophosphorylation. an inhibitory signal which is thought to provide an We first examined whether p110 alone was able to important negative feedback mechanism for antibody induce Btk autophosphorylation using the fibroblast co- production. The FcγRIIb1 inhibitory signal is known to expression system in which our previous studies were cause a block in sustained calcium signaling due to its performed (Figure 5A, top left panel). p110 induces an apparent ability to specifically block calcium influx easily detectable enhancement of wild-type Btk tyrosine (Bijsterbosch and Klaus, 1985; Amigorena et al., 1992; phosphorylation which is entirely dependent on Btk activ- Muta et al., 1994), and recently has been shown to ity. We then investigated whether Src kinases could require the SHIP inositol phosphatase, an enzyme with synergize with p110 in inducing Btk phosphorylation the in vitro capability to degrade PtdIns-3,4,5-P (Gupta (Figure 5A, top right panel) and whether Src kinase- et al., 1997; Ono et al., 1997). Since PtdIns-3,4,5-P / induced Btk phosphorylation was wortmannin inhibitable Tec kinase-dependent calcium signals are sustained as (bottom left panel). Co-expression of Lyn and Btk or of the result of enhanced calcium influx (Fluckiger et al., p110 and Btk both induce Btk tyrosine phosphorylation, 1998), we examined how co-engagement of the BCR and expression of all three produces a further enhancement and FcγRIIb1 affected PtdIns-3,4,5-P levels and Tec of this. In addition, treatment of cells expressing Lyn and kinase-dependent signaling (Figure 6). We first compared Btk with 100 nM wortmannin substantially inhibits the the levels of PtdIns-3,4,5-P observed after either BCR Lyn-dependent Btk tyrosine phosphorylation. Alterations engagement alone or co-engagement with FcγRIIb1 in Lyn activity (as assessed by its ability to induce tyrosine (Figure 6A). While BCR engagement resulted in a phosphorylation of the Syk tyrosine kinase under identical sustained PtdIns-3,4,5-P signal, no PtdIns-3,4,5-P was 3 3 conditions) by wortmannin or p110 expression do not detectable in this experiment at any time point after BCR– occur (data not shown). In further support of the idea that FcγRIIb1 co-engagement, although in other experiments p110 acts in concert with Src kinases, p110γ produces a small amounts of PtdIns-3,4,5-P could be detected at similar enhancement of Btk tyrosine phosphorylation only early time points only (data not shown). We then when it is co-expressed with Btk in a rat fibroblast line examined the effect of BCR–FcγRIIb1 co-engagement which constitutively expresses an activated Src mutant on Tec kinase-dependent calcium signaling and PLCγ2 (srcE387G) (Li et al., 1997). Identical p110-dependent tyrosine phosphorylation. In both control and Btk- enhancement of Btk tyrosine phosphorylation also occurs overexpressing B cells, FcγRIIb1 was able to inhibit in the A20 B-cell environment when Btk and p110 are sustained calcium signals (Figure 6B) and Btk-dependent co-expressed (Figure 5A, bottom right panel), and is PLCγ2 tyrosine phosphorylation (Figure 6C). Similar wortmannin inhibitable (data not shown). These results results for the calcium signal were obtained when other closely parallel the observed Btk-dependent PLCγ2 tyros- Tec kinases were expressed (data not shown). Strikingly, ine phosphorylation and IP production, consistent with analysis of Btk tyrosine phosphorylation in both control our previous findings that Btk autophosphorylation is and Btk-overexpressing B cells showed no differences required for its activation (Rawlings et al., 1996). in Btk tyrosine phosphorylation after either BCR 1967 A.M.Scharenberg et al. Fig. 5. PtdIns-3,4,5-P interaction with the Btk PH domain induces Btk autophosphorylation. (A) Active p110 is able to induce Btk autophosphorylation. Top left and top right panels: NIH 3T3 fibroblasts were infected for 6 h with the indicated vaccinia viruses, harvested and resuspended at 10 cells/ml in calcium buffer. Cells were then quickly spun down and lysed, and post-nuclear supernatants were immunoprecipitated with the indicated antibodies or analyzed directly. Bottom left panel: NIH 3T3 cells were infected as indicated, and 100 nM wortmannin was added as indicated to the infection media for the last1hof infection. Cells were then processed in the same fashion as in the top panels. Bottom right panel: A20 B cells were infected for 14 h with the indicated vaccinia viruses and then harvested, stimulated and lysed as in Figure 4. Post-nuclear supernatants subsequently were immunoprecipitated with the indicated antibodies or analyzed directly. (B) p110-dependent Btk phosphorylation requires kinase activity and an intact PH domain but not an SH2 domain. Top panel: A20 B cells were infected for 14 h with the indicated vaccinia viruses and then otherwise analyzed as in the bottom right panel of (A). Bottom panel: NIH 3T3 cells were infected for 6 h with the indicated viruses, and then otherwise analyzed as in the top left panel of (A). engagement alone or BCR–FcγRIIb1 co-engagement examined the effect of SHIP expression on both Lyn- (data not shown). We therefore reasoned that perhaps induced and Lyn–p110-induced tyrosine phosphorylation only a small fraction of the total Tec kinases in the of Btk in NIH 3T3 cells. Co-expression of SHIP, but cells was participating in the receptor-induced signaling not a catalytically inactive SHIP mutant, was able events. In order to produce an environment in which to block Lyn-induced Btk tyrosine phosphorylation the high local concentration of signaling proteins likely (Figure 6D, left), and Lyn–p110-induced Btk phospho- to be present in BCR–FcγRIIb1 complexes would be rylation (Figure 6D, right top). The expression of SHIP able to affect the majority of the expressed Btk, we was also associated with a complete loss of the p110- 1968 PIP /Tec kinase calcium signaling pathway dependent PtdIns-3,4,5-P signal, confirming the primary activated by an upstream kinase. In the case of Akt, role of PtdIns-3,4,5-P among other D3 phosphoinositides PtdIns-3,4-P interacts with the Akt PH domain and 3 2 in Tec kinase activation (Figure 6D, right bottom). induces its partial activation. This mechanism is enhanced further by a second serine/threonine kinase, PDK-1, which is able to phosphorylate Akt and lock it into a fully active Discussion state. In the case of Btk, the prototype Tec kinase, We have presented genetic, pharmacological and biochem- interaction of its PH domain with PtdIns-3,4,5-P is ical evidence that PtdIns-3,4,5-P initiates Tec kinase sufficient to produce Btk autophosphorylation in a cellular activation and subsequent PLCγ2 tyrosine phosphorylation context. This effect is enhanced further by Src family and IP production. Our results provide a mechanistic kinases, at least in part through their ability to phosphoryl- link between D3 phosphoinositides and regulation of ate Btk within its activation loop (Rawlings et al., 1996). intracellular calcium levels, and represent only the second Therefore, while interacting with distinct effector proteins example of a cellular process which has been shown to in the context of quite different cellular processes, PtdIns- be controlled directly through an interaction with a D3 3,4-P and PtdIns-3,4,5-P appear to utilize functionally 2 3 phosphoinositide, the other being the activation of the Akt equivalent mechanisms in which they promote the mem- serine/threonine kinase during its role in anti-apoptotic brane association of their respective target kinase for the signaling (reviewed in Toker and Cantley, 1997). The purpose of promoting its activation by a second kinase. mechanisms through which Akt and Tec kinases are Taking into account what has been shown previously activated by interaction with D3 phosphoinositides appear regarding the mechanism of Btk activation, a complete to be strikingly analogous: in each case, phosphoinositide molecular mechanism for a Tec kinase calcium signaling binding to the PH domain mediates their ability to be pathway emerges based on our data: PtdIns-3,4,5-P initi- ates Tec kinase activation in concert with Src kinases, as well as probably targeting the kinase to the plasma membrane. Once the Tec kinase is bound to PtdIns-3,4,5- P and activated, an interaction between its SH2 domain and a tyrosine-phosphorylated ligand induced by BCR engagement is required to co-localize the activated Tec kinase and PLCγ2 for the purpose of tyrosine phosphorylat- ing PLCγ2. The importance of the latter interaction is supported by (i) the normal to enhanced PtdIns-3,4,5-P - induced activation of the Btk-R307K SH2 mutant coupled with its inability to participate in BCR-mediated PLCγ2 tyrosine phosphorylation; and (ii) our previous results indicating that the Btk SH2 domain is required for Btk (activated by co-expression with Lyn) to phosphorylate Fig. 6. SHIP-dependent PtdIns-3,4,5-P degradation blocks PtdIns- 3,4,5-P /Tec kinase-dependent signaling. (A) Co-engagement of the BCR and FcγRIIb1 blocks BCR-induced PtdIns-3,4,5-P accumulation. Uninfected A20 B cells were labeled with P and prepared for stimulation as above, then stimulated with either 15 μg/ml of Fab rabbit anti-mouse IgG or 30 μg/ml of intact rabbit anti-mouse IgG, followed by extraction and analysis of lipids as in Figure 3 above. Similar results were obtained with infected cells, although the magnitude of the Fab -stimulated PtdIns-3,4,5-P production was 2 3 reduced. (B) Co-engagement of the BCR and FcγRIIb1 blocks Tec kinase-dependent calcium signaling. A20 B cells were infected either with control virus or Btk, loaded with fura-2, and prepared for calcium assays as described above. Cells were then stimulated with either 15 μg/ml Fab or 30 μg/ml rabbit anti-mouse IgG, while fura-2 fluorescence was monitored by bulk spectrofluorimetry. (C) Co- engagement of the BCR and FcγRIIb1 blocks Tec kinase-dependent phosphorylation of PLCγ2. A20 B cells were infected either with control virus or Btk, stimulated with either 15 μg/ml Fab or 30 μg/ ml intact rabbit anti-mouse IgG, and PLCγ2 tyrosine phosphorylation was analyzed as described above. Identical results were obtained when stimulations were performed with intact rat anti-mouse IgG with or without pre-treatment with 2.4G2 (to block binding of the Fc fragment of intact rat IgG with the inhibitory Fc receptors), demonstrating that the differences in observed PLCγ2 tyrosine phosphorylation are not due to differences in the activation of the BCR. (D) SHIP blocks Lyn- and Lyn–p110-dependent activation of Btk. Left panel: Btk was expressed alone or with the indicated viruses, followed by analysis of phosphotyrosine content as in Figure 5. Right panel: Lyn, Btk and p110 were expressed with SHIP or with a catalytically inactive form of SHIP in NIH 3T3 cells. Each sample was divided into two parts. One part was used for analysis of Btk phosphotyrosine content as in Figure 5 (top), while the second part was used for lipid extraction and analysis of the level of PtdIns-3,4,5-P as in (A) (bottom). 1969 A.M.Scharenberg et al. PLCγ2 when they are co-expressed in fibroblasts in the the effect of FcγRIIb1 was first described. While FcγRIIb1 absence of receptor stimulation (Fluckiger et al., 1998). is known to recruit SHIP, it has been unclear whether SHIP- In the case of BCR-mediated signals, candidate Tec SH2 mediated breakdown of IP (which has been linked to cal- ligands would be the Syk tyrosine kinase, which is also cium influx in some cell types) or PtdIns-3,4,5-P is respons- required for BCR-mediated calcium mobilization, and ible for the block in sustained calcium signaling. The results which is capable of binding both PLCγ2 and Btk in vitro presented here demonstrate that engagement of FcγRIIb1 is (Takata et al., 1994; Law et al., 1996; Wan et al., 1997), associated with a lack of accumulation of PtdIns-3,4,5-P , or PLCγ2 itself. In other systems, other receptor-associated inhibition of Tec kinase-dependent PLCγ2 tyrosine kinases or a structural feature of the receptor itself might phosphorylation and inhibition of Tec kinase-dependent cal- provide the co-localizing signal. This mechanism provides cium signaling, and that SHIP is able to degrade PtdIns- a full molecular framework with which to predict how 3,4,5-P and block Tec kinase activation in a heterologous mutations in Btk which cause X-linked agammaglobuline- system. Together, these data suggest that SHIP-dependent mia are able to interrupt signaling: PH mutations block Btk degradation of PtdIns-3,4,5-P in the local neighborhood membrane targeting and all subsequent events including its of BCR signaling complexes blocks calcium signaling by activation, SH2 mutations block the ability of membrane producing local deactivation of Tec kinases and consequent targeted/activated Btk to co-localize with PLCγ2 or other decreased PLCγ2 activation, as well as loss of the cofactor/ substrates, and kinase domain mutations block the ability substrate access functions of PtdIns-3,4,5-P (as discussed of membrane targeted/co-localized Btk to tyrosine phos- above). As demonstrated in Fluckiger et al. (1998), the phorylate PLCγ2 or other substrates. resulting inhibition of IP production allows an initial endo- The ability of PtdIns-3,4,5-P to initiate Tec-dependent plasmic reticulum (ER) calcium release followed by ER PLCγ2 tyrosine phosphorylation provides at least a partial calcium store refilling and so a loss of the store-operated molecular explanation for previous observations that wort- calcium influx required for sustained calcium signals, mannin is able to inhibit BCR-mediated IP production and accounting for the observed apparent selectivity of FcγRIIb1 calcium mobilization (Hippen et al., 1997; Kiener et al., for blocking calcium influx. 1997). In addition, our data further suggest that D3 phospho- In summary, our data implicate PtdIns-3,4,5-P as a inositides may play a second role in the regulation of PLCγ2, critical regulator of calcium signaling at least in part either alone or with Tec kinases. Comparison of the effect through its ability to initiate Tec kinase activation and of wortmannin on PLCγ2 tyrosine phosphorylation resulting tyrosine phosphorylation of PLCγ2 and IP (Figure 5C, compare lanes 2 and 8) and calcium fluxes production. A role for PtdIns-3,4,5-P –PH domain inter- (Figure 2A and B) in cells expressing Btk alone but not actions in Tec kinase-dependent PLCγ activation provides treated with wortmannin with those in cells co-expressing a molecular basis for why mutations which eliminate this p110–Btk but treated with wortmannin, shows similar levels interaction in the B cell-specific Tec member Btk cause of PLCγ2 tyrosine phosphorylation but markedly different aberrant B-cell development in humans and mice, and a sustained levels of intracellular calcium. This implies that molecular explanation for the inhibition of calcium in addition to tyrosine phosphorylation, PLCγ2 requires signaling mediated by the association of the SHIP inositol another factor(s) to be present for it to hydrolyze its PtdIns- 5-phosphatase with the inhibitory receptor FcγRIIb1. As 4,5-P substrate to IP effectively, a finding consistent with exemplified by BCR–FcγRIIb1 co-engagement, the PtdIns- 2 3 the normal levels of BCR-induced PLCγ2 tyrosine phospho- 3,4,5-P /Tec kinase calcium signaling pathway provides a rylation found in XLA-derived B-cell lines (Fluckiger et al., mechanism for cells to control the magnitude of calcium 1998). Assuming that wortmannin is only blocking D3 phos- signals initiated by receptors which utilize PLCγ isozymes phoinositide production, this second factor may involve independently of the strength of the external stimulus to direct regulation of PLCγ1/2 by PtdIns-3,4,5-P , as has been those receptors, and may explain the apparent involvement suggested for PLCγ1 (Rhee and Bae, 1997; Falasca et al., of PI3K/Tec signaling in signaling through co-stimulatory 1998). However, we favor the possibility that the second receptors such as CD19 or CD28. The tissue distribution factor involves PtdIns-3,4,5-P acting together with Tec of Tec kinases, the diversity of receptors capable of kinases independently of their activity based on the follow- activating them, and the recent appreciation of the signi- ing: (i) the relatively small effect of overexpression of p110 ficance of receptor-mediated inhibitory signals involving alone on IP and calcium (see Figure 1); (ii) the lack of inositol 5-phosphatases (Scharenberg and Kinet, 1996) dominant-negative effect on IP and calcium when inactive suggest that the PtdIns-3,4,5-P /Tec signaling mechanism 3 3 Btk is overexpressed (see Figure 2); and (iii) a Btk-deficient is likely to be of broad importance. form of the DT-40 cell line which produces no calcium signal, but regains a partial signal when it is re-transfected Materials and methods with inactive Btk (Takata and Kurosaki, 1996). A potential unifying explanation for these data is that PtdIns-3,4,5-P Cell culture, transformation assays, recombinant virus production and cDNAs functions as an inducible ‘tag’ for caveolae islands of A20 B cells were grown in RPMI-1640 with 10% fetal bovine serum PtdIns-4,5-P (Pike and Casey, 1996); and that PtdIns-3,4,5- 2 –5 and 10 M 2-mercaptoethanol. NIH 3T3 cells were grown in Dulbecco’s P /Tec kinase complexes function as adaptors to bring modified Eagle’s medium (DMEM) with 10% calf serum. PtdIns-4,5-P into proximity with activated PLCγ2. Identi- A20 infections were performed by adding 5 p.f.u./cell of recombinant virus to ~20% confluent A20 cells and allowing infections to proceed fying which of the above scenarios best explains PLCγ1/2 for 12–15 h. Where appropriate, control recombinant virus was added function is an important goal for future investigations. so that all samples were exposed to an equal number of p.f.u./cell. A The mechanism by which engagement of FcγRIIb1 with recombinant virus containing a cDNA encoding human Gβ1 inserted in the BCR or other antigen receptor is able to inhibit sustained an antisense orientation was used as the control virus because the calcium signals has been the subject of much interest since transcript generated was similar in length to that of Btk. 1970 PIP /Tec kinase calcium signaling pathway The cDNAs for p110α, Tec, wild-type Btk and all mutant forms of functions of IgG Fc receptors in B lymphocytes. Science, 256, Btk have been described previously (Hiles et al., 1992; Rawlings et al., 1808–1812. 1996; Fluckiger et al., 1998). The cDNA for the p110* construct has Andreotti,A.H., Bunnell,S.C., Feng,S., Berg,L.J. and Schreiber,S.L. also been described previously (Hu et al., 1995). (1997) Regulatory intramolecular association in a tyrosine kinase of The retrovirus expressing p110* was constructed by subcloning the the Tec family. Nature, 385, 93–97. p110* cDNA into the pSRαMSVtk-neo retroviral recombination vector Auger,K.R., Serunian,L.A., Soltoff,S.P., Libby,P. and Cantley,L.C. (1989) and selecting for recombinant retroviruses. Procedures used for the PDGF-dependent tyrosine phosphorylation stimulates production of production of retroviruses for transformation assays have been described, novel polyphosphoinositides in intact cells. Cell, 57, 167–175. as has the recombinant Btk retrovirus and the procedures used for the Bijsterbosch,M.K. and Klaus,G.G.B. (1985) Crosslinking of surface transformation assays (Afar et al., 1996). immunoglobulin and Fc receptors on B lymphocytes inhibits The p110-expressing recombinant vaccinia virus was constructed by stimulation of inositol phospholipid breakdown via the antigen subcloning a p110α cDNA into the pSC-66 vaccinia recombination receptors. J. Exp. Med., 162, 1825–1836. plasmid. Recombinant viruses were then selected and amplified using Bunnell,S.C., Henry,P.A., Kolluri,R., Kirchhausen,T., Rickles,R.J. and standard techniques (Earl et al., 1987). All recombinant Btk vaccinia Berg,L.J. (1996) Identification of Itk/Tsk Src homology 3 domain viruses have been described previously (Rawlings et al., 1996). ligands. J. Biol. Chem., 271, 25646–25656. Carpenter,C.L, and Cantley,L.C. (1996) Phosphoinositide kinases. Curr. Opin. Cell Biol., 8, 153–158. Pharmacologic reagents, antibodies, cell lysis, Conley,M.E. and Rohrer,J. (1995) The spectrum of mutations in Btk that immunoprecipitations and Western blotting cause X-linked agammaglobulinemia. Clin. Immunol. Immunopathol., Anti-phosphotyrosine antibody 4G10 was obtained from Upstate Biotech- 76, S192–S197. nology. Polyclonal anti-Btk has been described previously (Rawlings Cory,G.O., Lovering,R.C., Hinshelwood,S., MacCarthy-Morrogh,L., et al., 1996). Polyclonal anti-PLCγ2 was obtained from Santa Cruz Levinsky,R.J. and Kinnon,C. (1995) The protein product of the c-cbl Biotechnologies. Stimulations of A20 B cells were performed with Fab protooncogene is phosphorylated after B cell receptor stimulation and rabbit anti-mouse IgG (15 μg/ml) or intact rabbit anti-mouse IgG (30 binds the SH3 domain of Bruton’s tyrosine kinase. J. Exp. Med., 182, μg/ml) purchased from Jackson Immunoresearch. Wortmannin was 611–615. obtained from Sigma. Desiderio,S. and Siliciano,J.D. (1994) The Itk/Btk/Tec family of protein- Cells were lysed in borate-buffered saline (pH 8.0) containing 0.5% tyrosine kinases. Chem. Immunol., 59, 191–210. Triton X-100, 2 mM Na vanadate, 5 mM EDTA, 5 mM NaF and 5 μg/ Earl,P.L., Cooper,N. and Moss,B. (1987) Expression of Proteins in ml of leupeptin, aprotinin and pepstatin. Immunoprecipitations, Western Mammalian Cells Using Recombinant Vaccinia Viruses. 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Journal

The EMBO Journal – Springer Journals

Published: Apr 1, 1998

Keywords: B‐cells; inositol trisphosphate; phospholipase C; receptor; tyrosine kinases

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