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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 35, Issue of August 27, pp. 25051–25060, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. phox Tetratricopeptide Repeat (TPR) Motifs of p67 Participate in Interaction with the Small GTPase Rac and Activation of the Phagocyte NADPH Oxidase* (Received for publication, March 5, 1999, and in revised form, May 11, 1999) Hirofumi Koga‡§, Hiroaki Terasawa¶i, Hiroyuki Nunoi**, Koichiro Takeshige§, Fuyuhiko Inagaki¶i, and Hideki Sumimoto‡§ ‡‡ From the ‡Department of Molecular and Structural Biology, Kyushu University Graduate School of Medical Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, the §Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-8582, Japan, the ¶Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan, iCREST, Japan Science and Technology Corporation (JST), and the **Department of Pediatrics, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan The small GTPase Rac functions as a molecular switch motif within the N-terminal regulatory region of PAK, a motif that is present in a variety of targets of Rac and Cdc42 (4). in several important cellular events including cytoskel- etal reorganization and activation of the phagocyte Although more than 10 targets of Rac have been discovered (1), NADPH oxidase, the latter of which leads to production molecular natures of the interactions, except the CRIB motif, of superoxide, a precursor of microbicidal oxidants. remain largely unknown. It is thus considered important to During formation of the active oxidase complex at the study Rac-target interactions especially in functionally well membrane, the GTP-bound Rac appears to interact with defined systems. phox the N-terminal region of p67 , another indispensable The phagocyte NADPH oxidase, dormant in resting cells, is activator that translocates from the cytosol upon phag- activated during phagocytosis to produce superoxide, a precur- phox ocyte stimulation. Here we show that the p67 N ter- sor of microbicidal oxidants (5– 8). The significance of the en- minus lacks the CRIB motif, a well known Rac target, zyme in host defense is indicated by chronic granulomatous but contains four tetratricopeptide repeat (TPR) motifs disease (CGD) patients suffering from recurrent severe infec- with highly a-helical structure. Disruption of any of the tion caused by defect of the superoxide producing activity (7, 8). N-terminal three TPRs, but the last one, results in de- Although the NADPH oxidase is originally discovered in phago- fective interaction with Rac, while all the four are re- cytes because of its abundance, it has recently been proposed quired for the NADPH oxidase activation. We also find that the enzyme is expressed in a variety of cells and reactive that Arg-102 in the third repeat is likely involved in oxygen species derived from superoxide that play a role in binding to Rac via an ionic interaction, and that replace- several signal transduction systems (6). The redox core of the ment of this residue with Glu completely abrogates the oxidase is a membrane-spanning flavocytochrome, cytochrome capability of activating the oxidase both in vivo and in phox phox phox , comprising the two subunits gp91 and p22 . Upon vitro. Thus the TPR motifs of p67 are packed to func- 558 phox tion as a Rac target, thereby playing a crucial role in the cell stimulation three cytosolic proteins, namely p47 , phox active oxidase complex formation. p67 , and Rac, translocate to membranes, where they inter- act with the cytochrome to form an active oxidase complex. All the five polypeptides are required for activation of the NADPH Rac1 and Rac2, members of the Rho family of small GTPases, oxidase in vitro, and CGD is caused by defect of any of the genes play a pivotal role in several important cellular functions in- encoding these proteins except Rac (5– 8). cluding cytoskeletal reorganization, gene expression, and acti- In assembly and activation of the phagocyte NADPH oxi- vation of the phagocyte NADPH oxidase following microbial dase, protein-protein interactions between the oxidase factors phox phox infection (1, 2). Rac serves as a molecular switch cycling be- play a crucial role (5, 9, 10). Both p47 and p67 harbor tween an active GTP-bound and an inactive GDP-bound states. two SH3 domains, which mediate specific interactions between phox In the active state, Rac interacts with a variety of target (ef- the factors: the C-terminal SH3 domain of p67 interacts phox phox fector) proteins to elicit cellular responses (1, 2). For example, with p47 , while the N-terminal one of p47 does with phox the protein kinase PAK is activated by interacting with Rac in p22 (11–15). At least two events elicited during intracellu- a GTP-dependent manner (3). This interaction is mediated via lar signal transduction in stimulated cells appear to function as binding of Rac to a Cdc42/Rac interactive binding (CRIB) a switch of the oxidase activation. One of the two is a confor- phox mational change of p47 : the N-terminal SH3 domain of phox p47 is normally inaccessible, and, upon cell stimulation, * This work was supported in part by grants from the Ministry of phox Education, Science, Sports, and Culture of Japan, the Uehara Memorial becomes unmasked to interact with p22 , an induced inter- Foundation, the Kato Memorial Bioscience Foundation, the Fukuoka action that is required for the oxidase activation (12, 15, 16). Cancer Society, and CREST (Core Research for Evolutional Science and The other critical event seems to be conversion of Rac to the Technology) of Japan Science and Technology Corp. (JST). The costs of active state: only the GTP-bound Rac, but not the GDP-bound publication of this article were defrayed in part by the payment of page one, activates the oxidase under cell-free conditions (16 –19), charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and introduction of Rac antisense oligonucleotides or expres- ‡‡ To whom correspondence should be addressed: Dept. of Molecular and Structural Biology, Kyushu University Graduate School of Medical Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: CGD, chronic granulomatous disease; TPR, tetratricopeptide repeat; 81-92-642-6213; Fax: 81-92-642-6215; E-mail: [email protected]. CD, circular dichroism; GST, glutathione S-transferase; PAGE, poly- kyushu-u.ac.jp. acrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; GTPgS, guanosine 59-3-O-(thio)-triphosphate. The abbreviations used are: CRIB, Cdc42/Rac interactive binding; This paper is available on line at http://www.jbc.org 25051 This is an Open Access article under the CC BY license. phox TPR Domain of p67 25052 as a Target for Rac GTPase mid pProEX-HTb expression vector (Life Technologies, Inc.). The DNA sion of a dominant negative form of Rac2 (T17N) inhibits su- phox fragments encoding p67 and its mutants were subcloned into the peroxide production in stimulated cells (20, 21). Rac1 in the pGEX-2T expression vector (Amersham Pharmacia Biotech). (His) - GTP-bound state can directly interact with the N-terminal tagged or GST fusion proteins were expressed in the Escherichia coli phox region of p67 , comprising approximately 200 amino acid strain BL21DE3 (Novagen) and purified by His-bind resin (Novagen) or residues (22). This region lacks a CRIB motif, an established glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), re- target of Rac (4), but appears to contain tetratricopeptide re- spectively, according to the manufacturer’s protocol. phox Various p67 fused to GST (10 mg) were transferred to a nitrocel- peat (TPR) motifs, as suggested solely by sequence alignment lulose filter using Hybri-slot (Life Technologies, Inc.) according to the with other proteins containing the motif (23, 24). Since Rac manufacturer’s protocol. The filter was incubated with the blocking proteins with a mutation leading to defective interaction with buffer (3% bovine serum albumin, 0.1% Triton X-100, 0.5 mM MgCl ,5 phox p67 are unable to activate the oxidase (22, 25–27), the mM dithiothreitol) for 2 h, and washed two times with buffer A (50 mM interaction is considered to be involved in the oxidase activa- Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl , 0.1 mM dithiothreitol). His- phox tion. The final conclusion that Rac-p67 interaction is re- tagged Rac2 (Q61L/C189S) (0.2 mg) was preloaded for 30 min at 30 °C with 2 mlof[g- P]GTP (NEN Life Science Products Inc; 6000 Ci/mmol, quired, however, has awaited studies using mutant proteins of phox 10 mCi/ml, 0.8 M) in the GTP-loading buffer (50 mM Tris-HCl pH 7.5, 5 the target p67 phox mM EDTA, 0.5 mg/ml bovine serum albumin). The freshly prepared Here we demonstrate that the p67 N-terminal region of probe was incubated for 5 min at room temperature with the GST fusion about 200 residues is not only sufficient but also required for proteins on the filter in buffer A containing 1 mM GTP and 1 mg/ml fully interacting with Rac. Circular dichroism (CD) spectrum of bovine serum albumin. The filter was washed 3 times with an ice-cold the region reveals that it contains highly a-helical structure, washing buffer (20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20). phox After the filter was dried up, it was exposed to a Fuji Imaging plate and comparison between human and mouse p67 supports (Fuji Photo Co.), and signals were detected with the image scanner the idea that the required region contains four TPR motifs, the STORM (Molecular Dynamics). first three of which are tandemly arranged. TPR motifs, each Cell-free Activation of the Phagocyte NADPH Oxidase—The mem- comprising a pair of antiparallel a-helices (24), are initially brane fraction of human neutrophils was prepared as described previ- phox identified as a tandemly repeated degenerate 34-amino acid ously (12). The DNA fragment encoding p47 was subcloned into the sequence in the nuclear protein Nuc2p (28) and the cell cycle (His) -tagged fusion protein plasmid pET-28a(1) expression vector (No- vagen). The fusion proteins were expressed in E. coli strain BL21DE3 division genes cdc16, cdc13, and cdc27 (29, 30). It is now real- (Novagen) and purified by His-bind resin (Novagen), according to the ized that the motif occurs in a wide variety of proteins present manufacturer’s protocol. The neutrophil membrane (17.5 mg/ml) was in organisms as diverse as bacteria, archaea, and eukarya (31, mixed with His-tagged Rac2 (7.5 mg/ml) preloaded with 100 mM GTPgS, 32), and is involved in protein-protein and protein-lipid inter- phox His-tagged p47 (3.7 mg/ml), and the indicated concentration of GST- phox actions (33, 34). Little is, however, known about molecular p67 or its mutants, followed by incubation with an optimal concen- nature of TPR-mediated interactions. tration of SDS (100 mM) for 2.5 min at room temperature in potassium phosphate buffer (100 mM, pH 7.0) containing 75 mM cytochrome c,10 Based on the crystal structure of the TPRs of the protein mM FAD, 1.0 mM EGTA, 1.0 mM MgCl , and 1.0 mM NaN . The reaction phosphatase PP5 (24), we have introduced mutations that are 2 3 was initiated by addition of NADPH (250 mM) to the reaction mixture. expected either to disrupt or to unaffect packing of the TPR The production of superoxide was measured at the rate of superoxide phox helices of p67 . The present findings show that the N-termi- dismutase-inhibitable ferricytochrome c reduction at 550 –540 nm with nal three TPRs, but the last one, are packed to interact with a dual-wavelength spectrophotometer (Hitachi 557) (15, 16). phox phox Rac, and that Arg-102 in the third TPR is likely involved in Transfection of Wild-type and R102E Mutant p67 in gp91 and phox p47 -transduced K562 Cells—We used a retroviral vector system, binding to Rac via an ionic interaction. The results obtained phox pSXLC/pHa, that utilizes an internal ribosome entry site fragment of here also provide evidence that the interaction between p67 phox encephalomyocarditis virus (36), to transduce the gp91 gene into the and Rac is required for the NADPH oxidase activation both in phox phox leukemia cell line K562 that expresses p22 but not gp91 (37). vivo and in vitro. Although the fourth TPR is dispensable for phox Cells highly expressing gp91 were selected using FACS scan with the interaction, it appears to play a essential role in the oxidase the monoclonal antibody 7D5 to detect functional cytochrome b com- phox phox activation. prising the two subunits gp91 and p22 (38). A bicistronic retro- virus vector encoding a human multidrug resistance gene (MDR1) and EXPERIMENTAL PROCEDURES phox the p47 gene (pHa-MDR-IRES-p47) (39) were further transduced to phox phox Preparation of cDNAs of Mutant Rac2 and p67 —The DNA frag- the stably transduced gp91 -expressing K562 cells. The doubly trans- ments encoding various forms of human Rac2 were constructed by duced cells were selected with 4 ng/ml vincristine, expanded in a drug- polymerase chain reaction-mediated mutagenesis, all of which con- free medium, and used for the following experiments. tained the C189S substitution to avoid being modified by isoprenylation Complementary DNAs encoding the full-length of wild-type and mu- phox (35). Constitutively active and dominant negative forms of Rac2 carried tant p67 carring the R102E substitution were subcloned into the Q61L and T17N substitutions, respectively. Mutations in the effec- pREP10 (Invitrogen), which were transfected by electroporation to the phox phox tor loop (D38K and D38R substitutions) were introduced into the active K562 cells that stably express both gp91 and p47 . The K562 cells Rac2 to obtain Rac2 (D38K/Q61L) and Rac2 (D38R/Q61L). The DNA (2 3 10 cells/ml) were electroporated in the presence of 10 mgofthe phox phox fragments encoding mutant forms of p67 were also constructed by wild-type or mutant form of p67 plasmid DNA at 170 V, 960 micro- polymerase chain reaction-mediated site-directed mutagenesis. All the farads using Gene Pulser (Bio-Rad). At 48 h post-transfection, cells constructs were sequenced to confirm their identity. were selected for 5 days with 250 mg/ml hygromycin B. phox phox Interaction between Rac and p67 in the Yeast Two-hybrid Sys- Expression of Oxidase Factors in K562 Cells—For detection of p47 phox tem—In the yeast two-hybrid system to investigate interaction between and p22 , K562 cells were sonicated and the lysates were applied to phox Rac and p67 , we used yeast strains HF7c containing two GAL4- 10% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were inducible reporter genes, HIS3 and lacZ. The multiple cloning sites of transferred to a polyvinylidene difluoride membrane (Millipore), and pGBT9 (CLONTECH), containing the GAL4 DNA-binding domain, and probed with polyclonal antibodies raised against the C-terminal peptide phox phox pGADGH (CLONTECH), containing the GAL4 trans activation domain, of p47 and with an anti-p22 monoclonal antibody. phox were modified so that the inserts from glutathione S-transferase (GST) For detection of p67 , proteins were immunoprecipitated from the fusion protein plasmids pGEX-2T (Pharmacia) can be readily trans- K562 cell lysates (2 3 10 cells) with rabbit polyclonal antibodies raised phox ferred in correct orientation and reading frames, to obtain pGBT9g and against the C-terminal peptide of p67 and protein A-Sepharose pGADGHg (14). Yeast cells were co-transfected with pairs of two-hybrid (Pharmacia). After incubation for3hat4 °C,the beads were washed plasmids and selected by growth on medium lacking tryptophan and four times with ice-cold phosphate-buffered saline (137 mM NaCl, 2.7 leucine. Cells containing both plasmids were picked up and plated on a mM KCl, 4.3 mM Na HPO , and 1.4 mM KH PO , pH 7.0). Bound 2 4 2 4 histidine-lacking medium to test protein-protein interaction. proteins were resuspended in the SDS sample buffer, subjected to Overlay Assay to Detect Interactions between Purified Recombinant SDS-PAGE (10%), and transferred to a polyvinylidene difluoride mem- phox Proteins—The DNA fragment encoding a constitutively active form of brane (Millipore). The membrane was probed with an anti-p67 Rac2 (Q61L) was subcloned into the (His) -tagged fusion protein plas- monoclonal antibody (12). 6 phox TPR Domain of p67 as a Target for Rac GTPase 25053 Activation of the NADPH Oxidase in the Whole Cell System—Super- oxide production by the K562 cells expressing wild-type or mutant phox p67 was determined as superoxide dismutase-inhibitable chemilu- minescence detected with an enhancer-containing luminol-based detec- tion system (DIOGENES; National Diagnostics) as described by de Mendez et al. (37). After the selection, K562 cells (2 3 10 cells) were resuspended in 1 M Hepes, pH 7.4, 120 mM NaCl, 5 mM KCl, 5 mM ml of HBSS buffer (17 m glucose, 1 mM MgCl ,and1mM CaCl ). After the addition of the 2 2 ml), the cells were stimulated for enhanced luminol-based substrate (40 30 min at 37 °C with 200 ng/ml phorbol 12-myristate 13-acetate (PMA). The chemiluminescence was assayed using luminometer (Auto Lumat LB953; EG & G berthold). The reaction was stopped by the addition of superoxide dismutase (50 mg/ml). Circular Dichroism (CD) Spectra—GST-p67 (1–203) and GST-p67 (1–203, R102E) were expressed in the E. coli strain BL21DE3 (Nova- gen) and purified by glutathione-Sepharose 4B beads (Amersham Phar- macia Biotech), as described above. After thrombin digestion to remove the GST tag, the protein fragments, namely p67 (1–203) and p67 (1– 203, R102E), were purified on a Q-Sepharose (Amersham Pharmacia Biotech) and RESOURSE S (Amersham Pharmacia Biotech), and their purities were analyzed by 12% SDS-PAGE. The concentrations of the proteins used for these studies were 5 mM in 10 mM sodium phosphate, pH 6.4. CD measurements were performed with a Jasco J-725 spec- trometer using rectangular quarts cells of 0.2-cm path length at 20 °C. Far-UV CD spectra were the average of eight accumulations taken at 50 nm/min. Secondary structural components were calculated by the method of Yang et al. (40) using software supplied by Jasco, Inc. NMR Measurements—The proteins without tags, p67 (1–203) and p67 (1–203, R102E), were dissolved at concentrations of 1 mM in 50 mM sodium phosphate, 150 mM NaCl, and 10 mM dithiothreitol-d in 90% H O and 10% D O, and then adjusted to pH of 6.9 (direct pH meter 2 2 reading). Their H NMR spectra were recorded on a UNITY inova 500 spectrometer operating at H 500 MHz and 25 °C with a spectral width of 7000 Hz. Chemical shifts were referenced relative to the internal standard 2,2-dimethyl-2-silapentane-5-sulfonate. phox FIG.1. Interaction between Rac2 and p67 . A, interaction of phox Rac2 with the N-terminal region of p67 . The yeast reporter strain RESULTS HF7c was co-transfected with pairs of recombinant plasmids pGBT9 phox The N-terminal Region of p67 Is Both Required and Suf- and pGADGH, the former encoding a constitutively active form of Rac2 (Rac2 (Q61L)) fused to the GAL4 DNA-binding domain, and the latter ficient for Interaction with Rac—To explore the region of phox phox encoding various deletion mutants of p67 (numbers indicate amino p67 for binding to Rac2, we prepared a series of deletion acid residues from the first methionine) fused to the GAL4 transacti- phox mutants of p67 to use them for the yeast two-hybrid system. vation domain. Its histidine-independent growth was tested as de- A constitutively active form of Rac2, carrying the Q61L substi- scribed under “Experimental Procedures.” B, interaction of various phox phox tution, interacted with the full-length p67 (p67-F) (Fig. 1A), Rac2 with the N-terminal region of p67 . The yeast reporter strain HF7c was co-transfected with recombinant plasmids pGBT9 encoding which agrees with the result obtained by the yeast two-hybrid Rac2 carrying various mutations and pGADGH encoding p67-N (ami- system using a different reporter system (35). Two C-terminal no acid residues 1–242): Q61L, a constitutively active form of Rac2; phox deleted p67 , p67-N (amino acids 1–242) and p67 (1–203), D38K/Q61L and D38R/Q61L, proteins with additional D38K and fully interacted with Rac2 (Fig. 1A). The findings indicate that D38R substitutions, respectively; and T17N, a dominant negative phox form. Its histidine-independent growth was tested as described under the N-terminal region of p67 is sufficient for the interaction “Experimental Procedures.” with Rac, which is consistent with the results obtained from an in vitro binding assay using purified proteins (22). On the other phox phox hand, a dominant negative form of Rac2, namely Rac2 (T17N), p67 . To define residues of p67 involved in Rac binding, was incapable of interacting with p67-F, p67-N, or p67 (1–203) we substituted the neutral residue Gln for each of all eight Arg phox (Fig. 1B and data not shown), confirming that the GTP-bound residues that occur in the p67 N terminus (Fig. 2A). The phox Rac2, but not the GDP-bound one, binds to p67 R102Q substitution resulted in severely impaired interaction . Further deletion of p67 (1–203) from either its N or C terminus resulted with Rac2, while the R38Q or R77Q substitution led to a slight in complete loss of the interaction with Rac2 (Fig. 1A). These defect of the interaction (Fig. 2B). On the other hand, five other phox results suggest that the p67 N-terminal domain comprising mutant proteins carrying an Arg 3 Gln substitution at 62, 66, 155, 184, or 188 interacted with Rac2 as strongly as the wild- about 200 residues is both required and sufficient for binding to Rac2. type one did (Fig. 2B). When Rac1 was used instead of Rac2, the same results were Four TPR Motifs Occur in the Rac-binding Domain of phox phox obtained: GTP-bound Rac1 interacted with the N terminus of p67 —The binding experiment using p67 proteins with phox p67 substitution of Gln for Arg suggests that approximately 150 (data not shown). These Rac GTPases share 92% amino acid identity with the identical effector loop of amino acid residues from the N terminus play a more important role, since residues 32– 40. Some mutations in the loop region result in the substitution at 155, 184, or 188 did not affect the interac- phox impaired interaction with p67 (22, 27) as well as decreased tion with Rac2 (Fig. 2). A search of SwissPlot data base with this region by Blastp algorithm revealed a weak sequence ability to support the NADPH oxidase activation (22, 41). One such mutation is substitution of Asn, a neutral hydrophilic similarity (20 –30% identity) to regions of Ssn6p, a general residue, for Asp-38 (27). Replacement of this residue by basic transcriptional repressor in Saccharomyces cerevisiae (42), and ones (D38K and D38R) also abrogated the interaction with also, to a lesser extent, those of human CDC27 protein (43) and phox p67 yeast TOM70, the 70-kDa translocase of outer membrane in (Fig. 1B). These observations raise the possibility that Asp-38 may interact with a basic residue in the N terminus of mitochondria (44). The regions of these proteins are composed phox TPR Domain of p67 25054 as a Target for Rac GTPase FIG.2. Roles of Arg residues in interaction between Rac2 and phox p67 . A, schematic representation of the eight Arg residues in the N phox terminus of p67 . Arrowheads with the residue number indicate the position of the eight Arg residues contained in the N-terminal region of phox p67 . Each shaded box represents TPR motif. B, interaction of Rac2 phox with p67 carrying an Arg3 Gln mutation. The yeast reporter strain HF7c was co-transfected with recombinant plasmids pGBT9 encoding phox Rac2 (Q61L) and pGADGH encoding p67 carrying an Arg 3 Gln mutation. Its histidine-independent growth was tested as described under “Experimental Procedures.” phox of TPR motifs. The motif is a degenerate 34-amino acid se- FIG.3. TPR motifs in p67 . A, sequence alignment of the TPR phox quence identified in a wide variety of proteins, present in motifs of SSN6, CDC27, TOM70, and p67 . Consensus TPR motif residues are shown with black and shaded boxes for small and large tandem arrays of 3–16 motifs (24, 28, 29, 31). Although there hydrophobic residues, respectively. Small hydrophobic residues are exists no position characterized by an invariant residue, a commonly observed at positions 8, 20, and 27. Position 32 is frequently consensus sequence pattern of small and large hydrophobic proline (boxed), located at the C terminus of helix B, and large hydro- residues has been defined: small hydrophobic residues are com- phobic residues are also located at particular positions, especially 4, 17, phox and 24. B, the TPR motifs of human and mouse p67 . Indicated monly observed at positions 8, 20, and 27, while large ones are phox residues of mouse p67 (46) are different from those of human one. at 4, 17, and 24 (24, 31). Careful alignment of the N terminus Consensus positions in the TPR motif are shown with black and shaded phox of p67 suggests that the region comprises four copies of the boxes for small and large hydrophobic residues, respectively. TPR motif, although the first repeat contains only 31 residues (Fig. 3A), the possibility which is also pointed out by other phox investigators (23, 24). In all four motifs of p67 , there exist small hydrophobic residues at positions 8, 20, and 27, and large hydrophobic ones at 4, 17, and 24. In addition, like other TPR phox sequences, the N-terminal domain of p67 are quite hydro- philic as estimated from hydrophilicity/hydrophobicity plots (45). phox Further support for the identity of the p67 N-terminal region as a TPR domain came from comparison between human phox p67 and its mouse homologue, the sequence of which we phox have recently determined (46). Since mouse p67 not only phox interacts with human Rac2 but also can replace human p67 in a cell-free activation system of human NADPH oxidase (46), phox critical residues of p67 are likely conserved between mouse FIG.4. Far-UV CD spectrum for the N-terminal region of phox and human. Alignment of amino acid sequences of human and p67 (p67 (1–203)). A, p67 (1–203) was purified and analyzed on phox SDS-PAGE, as described under “Experimental Procedures.” Positions mouse p67 revealed that most of substitutions in the TPRs of molecular size standards are indicated to the right in kilodaltons. B, occur at nonconsensus positions; consensus residues are selec- the far-UV CD spectrum of p67 (1–203) (5 mM) was measured at 20 °C tively conserved between the two species (Fig. 3B). in 10 mM sodium phosphate, pH 6.4. To obtain direct information on the structure of the N termi- phox nus of p67 (residues 1–203) as a TPR domain, we isolated the fragment (Fig. 4A) and measured the circular dichroism ranged, while 16 extra residues are located between the third (CD) spectrum (Fig. 4B). The profile, with the maximum at 190 and fourth repeats (see Fig. 2A). phox nm and minima at 208 and 220 nm, is characteristic of an Role of p67 TPR Motifs in Binding to Rac—To clarify phox a-helix. The proportions of a-helix, b-sheet, and remaining roles for each TPR motif of p67 in binding to Rac, we structures were estimated by the method of Yang et al. (40) to introduced two types of systematic mutations that are expected be 76.3, 0, and 23.7%, respectively. This finding supports the to disrupt each TPR architecture, based on the crystal struc- phox idea that the N terminus of p67 contains TPR motifs, since ture of the TPR domain of the protein phosphatase PP5 (24). the motif comprises a pair of antiparallel a-helices (24). Taken Each of the three TPR motifs of this domain consists of a pair phox together, we concluded that the N-terminal region of p67 of antiparallel a-helices of equivalent length, termed helix A contains four TPR motifs, the first three being tandemly ar- and helix B (Fig. 3). Adjacent TPR motifs are packed together phox TPR Domain of p67 as a Target for Rac GTPase 25055 clude the possibility, we purified mutant proteins as GST fu- sions (Fig. 5B) and tested their ability to bind to Rac2 by an overlay assay. As shown in Fig. 5C, a negligible binding was observed using the proteins with a substitution in any of the N-terminal three TPRs, whereas GTP-bound Rac2 interacted phox well with the mutant p67 carrying the A128Q substitution. phox Thus the TPR motifs of p67 , except the last one, are likely involved in Rac interaction. Alternatively, one or more of the three TPRs may play a critical role in the correct overall folding of the TPR domain, which is required for binding to Rac2. Involvement of the second and third TPRs is also supported by the observation that the interaction with Rac2 is prevented by one amino acid substitution within helix A of these TPR (R38Q and R77Q) (Fig. 2). For the second type of mutation, we deleted a residue at position 22 in the TPR motifs (Asp-27, Lys-58, Lys-92, and Glu-142). The deletion of position 22 is expected to change the spacing between the conserved small residues (positions 20 and 27) within helix B, leading to the incorrect packing between adjacent TPRs (Ref. 24; for detail see “Discussion”). One of the phox deletions, K58D, occurs in a patient with CGD, whose p67 is relatively unstable and defective in binding to Rac (48). Exper- iments using the yeast two-hybrid system showed that Rac binding activities of proteins with the first to third TPR motif mutation (D27D, K58D, and K92D) were severely reduced or abolished, while that of the fourth TPR mutant (E142D) was preserved as well as the wild-type protein (Fig. 5A). The in vitro binding activities by the overlay assay using the purified mu- tant proteins (Fig. 5B) were consistent with those obtained by the yeast two-hybrid system (Fig. 5C), confirming a crucial role FIG.5. Interaction of Rac2 with two types of TPR mutants of phox phox of the first three TPRs. p67 . The first type of the mutations introduced into p67 (left) phox carries a substitution of the bulky residue Gln for a conserved small All TPR Motifs of p67 Are Required for Activation of the phox residue at position 8; G13Q, G44Q, G78Q, and A128Q. The second type NADPH Oxidase—The N terminus of p67 , p67 (1–242), is (right) mutation is deletion of an amino acid residue at position 22 in enough to fully activate the phagocyte NADPH oixdase in a the TPR motifs; D27D, K58D, K92D, and E142D. A, the yeast reporter cell-free system reconstituted with human neutrophil mem- strain HF7c was co-transfected with recombinant plasmids pGBT9 phox encoding Rac2 (Q61L) and pGADGH encoding various TPR mutants of brane, p47 , and Rac2 (16). To study the role of the TPR phox phox p67 . Its histidine-independent growth was tested as described under motifs of p67 in the oxidase activation, we prepared the “Experimental Procedures.” B, SDS-PAGE analysis of wild-type and protein lacking the first three or all TPR motifs, namely p67 phox TPR mutants of p67 . Each sample (0.1 mg) as GST fusion protein is (126 –242) or p67 (170 –242), respectively (Fig. 6A), and esti- resolved on a 10% SDS-PAGE and visualized with Coomassie Brilliant Blue. Lane 1, GST-p67-N; lane 2, GST-p67-N (G13Q); lane 3, GST- mated their abilities to activate the enzyme in the cell-free p67-N (G44Q); lane 4, GST-p67-N (G78Q); lane 5, GST-p67-N (A128Q); system (15, 16). Both proteins were incapable of supporting lane 6, GST-p67-N (D27D); lane 7, GST-p67-N (K58D); lane 8, GST- superoxide production under the cell-free conditions, even at 2 p67-N (K92D); and lane 9, GST-p67-N (E142D). Positions of molecular order higher concentrations than those for p67 (1–242), con- size standards are indicated to the left in kilodaltons. C, analysis of phox Rac2 binding activity of various TPR mutants of p67 by an overlay taining all the four motifs, to activate the oxidase (less than 1 phox assay. Each wild-type or mutant p67 (10 mg) as GST fusion protein mg/ml) (Fig. 6B). were put on a nitrocellulose filter, and probed with His-tagged Rac2 To estimate importance of each TPR motif, we next used the preloaded with [g- P]GTP. The filter was exposed to an imaging plate, phox p67 proteins carrying a mutation in one of the TPRs (Fig. which was subjected to the image scanner, as described under “Exper- imental Procedures.” 5). The proteins with a mutation in the first three TPRs were unable to activate the NADPH oxidase (Fig. 6C). The incapa- bility is likely due to that these mutant proteins are not able to in a parallel arrangement such that a tandem TPR motif struc- interact with Rac2 (Fig. 5). Intriguingly, the proteins carrying ture is composed of a regular series of antiparallel a-helices the A128Q substitution or the deletion of Glu-142 showed little (24). phox or no activity for the oxidase activation (Fig. 6C), although both The first type of the mutations introduced into p67 is mutant proteins fully interacted with Rac (Fig. 5). Thus the substitution of the bulky residue Gln for a conserved small fourth TPR plays an essential role in activation of the NADPH residue at position 8 (Gly-13, Gly-44, Gly-78, or Ala-128). Such oxidase, possibly interacting with other oxidase factors such as mutations are expected to cause the incorrect packing of neigh- phox cytochrome b or p47 . boring helices of a TPR (Ref. 24, for detail, see “Discussion”), phox Arg-102 in the Third TPR of p67 Is Involved in Rac and a similar mutation at this position of the third TPR of phox p67 (G78E) has been reported to cause CGD (47). The Binding, Probably via an Ionic Interaction—As shown above (Fig. 2), the replacement of the basic residue Arg-102 by the substitution in the second or third TPR (G44Q or G78Q, re- spectively) led to severely impaired two-hybrid interaction with neutral hydrophilic residue Gln resulted in severely defective interaction with Rac2. This residue, at position 32 of the third Rac2 (Fig. 5A). While the protein carrying the first TPR muta- phox tion (G13Q) weakly interacted with Rac2, the mutation in the TPR of p67 , is conserved between mouse and human (Fig. last TPR (A128Q) did not affect the interaction (Fig. 5A). It is 3B). The position is located at the C terminus of helix B, and possible that these substitutions may destabilize the proteins, thus is expected to be exposed but not involved in defining the thereby resulting in a loss of two-hybrid interactions. To ex- TPR architecture, as in the TPR domain of PP5 (24). Therefore phox TPR Domain of p67 25056 as a Target for Rac GTPase phox FIG.7. Effects of substitutions for Arg-102 in p67 on bind- phox ing to Rac2. A, Rac2 binding activities of p67 carrying various substitutions for Arg-102 were tested in the yeast two-hybrid system. The yeast reporter strain HF7c was co-transfected with recombinant plasmids pGBT9 encoding Rac2 (Q61L) and pGADGH encoding p67-N carrying various substitutions for Arg-102. Its histidine-independent growth was tested as described under “Experimental Procedures.” B, phox SDS-PAGE analysis of wild-type and various mutant forms of p67 . Each sample (0.4 mg) as GST fusion protein was resolved on a 10% SDS-PAGE and visualized with Coomassie Brilliant Blue. Lane 1, GST- p67-N; lane 2, GST-p67-N (R102K); lane 3, GST-p67-N (R102Q); and lane 4, GST-p67-N (R102E). Position of molecular size standards are phox FIG.6. Ability of various mutant p67 to activate the phag- indicated to the left in kilodaltons. C, analysis of Rac2 binding activity phox ocyte NADPH oxidase under cell-free conditions. A, SDS-PAGE of mutant p67 carrying substitutions for Arg-102 by an overlay phox analysis of p67-N (1–242) and its deletion mutants. Each sample (0.5 assay. The wild-type and mutant p67 as GST fusion proteins (10 mg) mg) as GST fusion protein was resolved on a 12% SDS-PAGE and were put on a nitrocellulose filter, and probed with His-tagged Rac2 visualized with Coomassie Brilliant Blue. Lane 1, GST-p67-N (1–242); preloaded with [g- P]GTP. The filter was exposed to an imaging plate, lane 2, GST-p67 (126 –242); and lane 3, GST-p67 (170 –242). Position of which was subjected to the image scanner, as described under “Exper- molecular size standards are indicated to the left in kilodaltons. B, imental Procedures.” human neutrophil NADPH oxidase was activated with the indicated concentration of the GST-p67-N or its deletion mutants, in the presence phox the estimated proportions of a-helix, b-sheet, and remaining of His-tagged p47 (3.74 mg/ml), His-tagged Rac2 (7.3 mg/ml), and human neutrophil membranes (17.5 mg/ml). Filled circles, open trian- structures in the mutated TPR domain were 76.9, 0, and 23.1%, gles, and open circles indicate superoxide producing activities using respectively. We also tested the stability of the proteins by p67-N (1–242), p67 (126 –242), and p67 (170 –242), respectively. Super- gradually increasing temperature from 20 to 60 °C: the oxide production was determined as described under “Experimental changes in helical content were monitored at 222 nm. The Procedures.” C, human neutrophil NADPH oxidase was activated with the wild-type or TPR mutants of GST-p67-N (10 mg/ml) under condi- curve for the changes of the R102E mutant protein was the tions as described in B. same as that of the wild-type one (data not shown), supporting phox the idea that the a-helices of the p67 TPR domain are not it is possible that Arg-102 conforms a binding surface and a disrupted by the substitution. positive charge of this residue mediates the interaction with Furthermore, and most importantly, little difference could be Rac. To test these possibilities, we introduced one amino acid observed between H NMR spectra of the wild-type and R102E phox substitution for Arg-102. The mutant p67 with the substi- protein (Fig. 8), indicating that the mutated TPR domain is tution of the positively charged residue Lys (R102K) could correctly folded. Thus the R102E substitution appears to unaf- interact with Rac2, but to a lesser extent, as assessed by the fect the structural integrity of the protein. Taken together with yeast two hybrid-system (Fig. 7A) as well as by an overlay the results obtained by the binding experiments, it is concluded phox assay using purified proteins (Fig. 7, B and C). On the other that Arg-102 of p67 is involved in binding to Rac, probably hand, the replacement by the neutral residue Ala or Leu via an ionic interaction. (R102A or R102L, respectively), like the R102Q substitution, Arg-102 Plays an Important Role in the Oxidase Activation in led to a severely defective interaction with Rac (Fig. 7). The Vitro—To elucidate the role of Arg-102 in the NADPH oxidase protein carrying the substitution of the acidic residue Glu activation, we tested the activity of mutant proteins carrying (R102E) could not bind to Rac2 at all (Fig. 7). various substitutions for Arg-102 under the cell-free conditions. To rule out the possibility that the R102E substitution re- The protein with substitution of the basic residue Lys (R102K) sults in a disrupted structure of the TPR domain, we measured was capable of supporting superoxide production, but to a both CD and H NMR spectra of the protein with this mutation. lesser extent than the wild-type one (Fig. 9). Replacement by The CD spectrum of the mutant protein (data not shown) was the neutral residue Gln (R102Q) or the acidic residue Glu in complete agreement with that of the wild-type one (Fig. 4B): (R102E) resulted in little or no activation of the NADPH oxi- phox TPR Domain of p67 as a Target for Rac GTPase 25057 FIG.8. Comparison of H NMR spectra between p67 (1–203) and p67 (1–203, R102E). 500 MHz H NMR spectra of p67 (1–203) (A) and p67 (1–203, R102E) (B) were measured in 50 mM sodium phos- phate, 150 mM NaCl, and 10 mM dithiothreitol-d in 90% H O and 10% 10 2 D O at 25 °C as described under “Experimental Procedures.” phox FIG. 10. Role of Arg-102 of p67 in the NADPH oxidase acti- phox phox vation in a whole cell system. A, expression of p67 in gp91 phox phox FIG.9. Ability of various mutant p67 with substitution for and p47 -transduced K562 cells. The doubly transduced K562 cells Arg-102 to activate the phagocyte NADPH oxidase under cell- were transfected with pREP10 vector or the vector to express the phox free conditions. Superoxide production was measured as described indicated form of p67 .Inthe upper panel, cell lysates were immu- phox under “Experimental Procedures” using the indicated concentration of noprecipitated with rabbit anti-p67 polyclonal antibodies, and the phox the wild-type or mutant GST-p67-N, His-tagged p47 (3.74 mg/ml), samples were resolved by SDS-PAGE, transferred to a polyvinylidene phox His-tagged Rac2 (7.3 mg/ml), and human neutrophil membranes (17.5 difluoride membrane, and immunoblotted with a mouse anti-p67 mg/ml). Open squares, filled circles, filled squares, and open circles monoclonal antibody. In the lower panel, cell lysates were immuno- phox indicate superoxide producing activities using p67-N (wild-type), p67-N blotted with rabbit anti-p47 polyclonal antibodies. B, PMA-induced phox phox (R102K), p67-N (R102Q), and p67-N (R102E), respectively. chemiluminescence by gp91 and p47 -transduced K562 cells phox transfected with the wild-type or R102E mutant of p67 . The K562 phox 6 cells expressing the indicated form of p67 (2 3 10 cells/ml) were dase, respectively (Fig. 9). The order of potency to activate the stimulated with PMA (200 ng/ml) and the chemiluminescence change was continuously monitored with an enhanced luminol-based sub- oxidase (the wild-type . R102K . R102Q . R102E) agrees strate, DIOGENES. Superoxide dismutase (SOD) (50 mg/ml) was added with that to bind to Rac (Fig. 7), providing strong evidence that phox phox where indicated. C, superoxide production by gp91 and p47 - phox oxidase activation requires the interaction between p67 and transduced K562 cells transfected with the wild-type or R102E mutant phox Rac. Thus activation of the NADPH oxidase likely involves an of p67 . Superoxide production is expressed as the percent activity phox relative to control cells transfected with wild-type p67 . Each graph ionic interaction with Rac via Arg-102 in the third TPR of phox represents the mean of data from seven independent transfections, with p67 , which is consistent with that this TPR plays a crucial bars representing the standard deviation of percent activity (n 5 7). role in the activation (Fig. 6C). phox p67 Carrying the R102E Substitution Is Incapable of the proteins. The transduced cells expressed functional cyto- Supporting the NADPH Oxidase Activation in a Whole Cell phox phox System—We finally investigated the role of Arg-102 in the chrome b comprising the two subunits gp91 and p22 phox NADPH oxidase activation in vivo, using a whole cell system of (data not shown; see “Experimental Procedures”) and p47 K562 cells, which is similar to the one that has been developed (Fig. 10). by Leto’s group (37). The cell line expresses Rac and low levels The doubly transduced K562 cells were subsequently trans- phox of endogenous p22 , but requires expression of the other fected with the episomal vector pREP10 that contained cDNA phox phox phox phox encoding the full-length wild-type p67 (p67-F) or full-length three oxidase factors (gp91 , p47 , and p67 ) to exhibit phox superoxide production in response to PMA (37). To explore the p67 with the R102E substitution, namely p67-F (R102E). phox phox function of p67 The wild-type p67 -expressing cells fully produced superox- , we transduced K562 cells for stable expres- phox phox sion of gp91 and p47 using retroviral vectors encoding ide when stimulated with PMA (Fig. 10). On the other hand, phox TPR Domain of p67 25058 as a Target for Rac GTPase the cells transfected with the p67-F (R102E) cDNA were unable face for Rac. Substitution of the basic residue Lys for Arg-102 to support superoxide production in response to PMA, although slightly reduces the capability of binding to Rac, while replace- the protein was expressed at a similar level as the wild-type ment by a neutral or acidic residue leads to little or no inter- phox phox p67 in the control cells (Fig. 10). Thus the mutant p67 action with Rac, respectively. Thus Arg-102 plays a crucial role with the R102E substitution is incapable of activating the in binding to Rac, probably via an ionic interaction. This may phagocyte NADPH oxidase under both cell-free and whole cell explain that replacement of Asp-38 in the effector loop of Rac by phox conditions. a neutral or basic residue abrogates binding to p67 (Fig. 1B; and Refs. 22 and 27). Taken together with the present experi- DISCUSSION ments using mutant proteins, the binding to Rac requires a phox phox Here we present that TPR motifs of p67 are involved in specific block of the TPRs of p67 , the first three motifs, the interaction with the small GTPase Rac, both structurally containing Arg-102 as an interacting residue. and functionally. The binding to Rac requires an overall struc- phox The TPRs of p67 by themselves, however, do not seem phox ture of the p67 N-terminal domain comprising about 200 sufficient for the interaction, since the protein fragment com- amino acid residues in the proper conformation. The domain prising the first three or all TPRs (p67 (1–122) or p67 (1–167), contains four TPR motifs, the N-terminal three being tandemly respectively) was incapable of binding to Rac2 (Fig. 1). A region arranged, while 16 extra residues are located between the third outside of the TPRs may be required for the structural integrity and fourth TPRs. The present results show that the first three of the TPR domain and/or for stable interaction between TPRs, but not the last one, play an essential role in the binding phox phox p67 and Rac. A recent report has shown that p67 amino to Rac, via directly interacting with the GTPase and/or via acid residues 170 –199 can bind to Rac, but to a much lesser being folded for the correct packing of the TPR domain. In extent (51). It can be excluded that the TPR motifs do not particular, the third TPR appears to be directly involved in the physically interact with Rac but provide the structural frame- interaction with Rac: Arg-102 in the third TPR, a residue that work to present residues 170 –199 effectively to Rac, because is likely irresponsible for the packing, participates in the inter- Arg-102 in the third TPR appears to directly bind to Rac: p67 action, probably via an ionic bond. (1–242, R102E), containing both residues 170 –199 and TPRs The structure of the TPR domain of the protein phosphatase with a mutation unaffecting the structural integrity, is incapa- PP5 reveals that each TPR motif of this domain consists of a ble of binding to Rac (Fig. 7). There may be two (or more) sites pair of antiparallel a-helices of equivalent length, helix A and phox of p67 that directly interact with Rac, both of which are helix B (24). Adjacent TPR motifs are packed together in a required for stable interaction and activation of the NADPH parallel arrangement such that a tandem TPR motif structure oxidase. The protein that contains residues 170 –199 but lacks is composed of a regular series of antiparallel a-helix: each the first three or all TPR motifs (p67 (126 –242) or p67 (170 – a-helix shares two immediate a-helix neighbors and the protein 242), respectively) is not capable of activating the oxidase at all, fold may be defined as an overlapping array of three-helix as shown in this study (Fig. 6B). bundles (24). Since a small residue at position 8 is located at phox Interaction of Rac with p67 has been considered to be the position of closest contact between the A and B a-helices of required for activation of the phagocyte NADPH oxidase, based a TPR (24), substitution of the residue for the bulky residue Gln on the observations that mutant forms of Rac, defective in the may lead to incorrect packing of the helix. This prediction is phox interaction, are incapable of activating the enzyme in vitro (22, supported by a mutation of the p67 gene in a patient with 25–27). The requirement, however, has not been evidenced by CGD: the mutant protein with substitution of position 8 in the phox experiments using mutant forms of the target protein p67 , third TPR (Gly-78) for Glu appears unstable in phagocytes (47), except a report showing that a protein containing deletion of probably due to misfolding of the TPR. In addition, mutations Lys-58, being unstable, neither binds to Rac nor activates the at this position within TPRs 5 and 7 of cdc23 result in defect of oxidase (48). The present study demonstrates that a series of protein function (49). Position 20 on helix B also resides be- phox TPR mutants of p67 , defective in Rac binding, were all tween both helices A and B, while position 27 is located at the devoid of activity in the cell-free activation system of the oxi- interface of three helices (A, B, and A9) within a three-helix phox dase (Fig. 6C). Among mutant proteins of p67 carrying bundle (24). This bundle may be incorrectly packed by one substitution for Arg-102, the Rac binding activity correlates amino acid deletion in the region of residues 21–26 within helix well with the capability of activating the oxidase in vitro (the B. Both types of mutations (substitution of Gln for a residue at wild-type . R102K . R102Q . R102E) (Fig. 9). Furthermore, position 8 and deletion of a residue at position 22) in the first to phox the protein with the R102E substitution, leading to a complete third TPRs of p67 result in defective interaction with Rac loss of interaction with Rac, is also inactive in the whole cell (Fig. 5). Thus the three TPRs are folded such that the TPR activation system of the oxidase (Fig. 10). These observations domain interacts with Rac. The conclusion can explain how phox provide strong evidence that the binding of Rac to p67 plays CGD is caused by three reported mutations within the first to phox an essential role in activation of the NADPH oxidase both in third TPRs of p67 : deletion of three amino acid residues vivo and in vitro. (Lys-19, Lys-20, and Asp-21) in the first TPR (50), deletion of phox On the other hand, the interaction between Rac and p67 Lys-58 in the second TPR (48) and substitution for Gly-78 in is not sufficient for activating the NADPH oxidase. The cor- the third TPR (47), the latter two of which are reported to result phox rectly packed fourth TPR of p67 , in contrast to the other in decreased amounts of the proteins in neutrophils (47, 48). TPRs, does not seem involved in the interaction (Fig. 5), but is Arg-102, on the other hand, resides at position 32 of the third required for activation of the NADPH oxidase (Fig. 6C). The TPR. Since the position is located at the C terminus of helix B (24), Arg-102 is not likely involved in the packing of the TPR fourth TPR may be packed independently of the N-terminal three TPRs; it is rather conformed together with other regions, helices. This is supported by the finding that the protein car- rying the R102E substitution appears to be as stable as the presumably forming an interface to interact with other oxidase phox phox phox factors, p47 or a cytochrome b subunit (gp91 or wild-type p67 in vivo (Fig. 10), and confirmed by the obser- phox p22 ). In this context, it should be noted that about 10 vations that substitution resulted in little change in both CD 1 phox (data not shown) and H NMR spectra (Fig. 8). This mutation residues C-terminal to the Rac-binding domain of p67 (res- phox idues 203–212) are also required for the oxidase activation (52, thus does not affect the structural integrity of p67 . The basic residue is rather considered to constitute a binding inter- 53). It has been shown that, in some proteins harboring mul- phox TPR Domain of p67 as a Target for Rac GTPase 25059 pHa. We also thank Dr. Futoshi Kuribayashi (Kyushu University) for technical advice, and Y. Kage (Kyushu University), E. Ebisui (Tokyo Metropolitan Institute of Medical Science), and Drs. M. Iwata (Kum- amoto University) and M. Y. Park (University of Tokyo) for technical assistance. REFERENCES 1. Van Aelst, L., and D’Souza-Schorey, C. (1997) Genes Dev. 11, 2295–2322 2. Hall, A. (1998) Science 279, 509 –514 3. 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(1997) FEBS Lett. 400, 136 –140 or related sequences (Fig. 11), in which one additional amino 34. Skinner, J., Sinclair, C., Romeo, C., Armstrong, D., Charbonneau, H., and acid residue is inserted between helices A and B when aligned Rossie, S. (1997) J. Biol. Chem. 272, 22464 –22471 35. Dorseuil, O., Reibel, L., Bokoch, G. M., Camonis, J., and Gacon, G. (1996) with TPRs. It has recently been shown that PRK2, a protein J. Biol. Chem. 271, 83– 88 kinase being considered as a target of Rho, can also interact 36. Sugimoto, Y., Aksentijevich, I., Gottesman, M. M., and Pastan, I. (1994) with Rac (58). Its Rac-binding site appears to reside in the HR1 Bio/Technology 12, 694 – 698 37. de Mendez, I., Adams, A. G., Sokolic, R. A., Malech, H. L., and Leto, T. L. (1996) region that contains three leucine zipper-like sequences (59). In EMBO J. 15, 1211–1220 regions overlapping the sequences, small and large hydropho- 38. Nakamura, M., Murakami, M., Koga, T., Tanaka, Y., and Minakami, S. 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Journal of Biological Chemistry – Unpaywall
Published: Aug 1, 1999