共查询到20条相似文献,搜索用时 46 毫秒
1.
Protein-tyrosine kinases and Rho GTPases regulate many cellular processes, including the reorganization and dynamics of the actin cytoskeleton. The Wiskott-Aldrich syndrome protein (WASP) and its homolog neuronal WASP (N-WASP) are effectors of the Rho GTPase Cdc42 and provide a direct link between activated membrane receptors and the actin cytoskeleton. WASP and N-WASP are also regulated by a large number of other activators, including protein-tyrosine kinases, phosphoinositides, and Src homology 3-containing adaptor proteins, and can therefore serve as signal integrators inside cells. Here we show that Cdc42 and the Src family kinase Lck cooperate at two levels to enhance WASP activation. First, autoinhibition in N-WASP decreases the efficiency (kcat/Km) of phosphorylation and dephosphorylation of the GTPase binding domain by 30- and 40-fold, respectively, and this effect is largely reversed by Cdc42. Second, Cdc42 and the Src homology 3-Src homology 2 module of Lck cooperatively stimulate the activity of phosphorylated WASP, with coupling energy of approximately 2.4 kcal/mol between the two activators. These combined effects provide mechanisms for high specificity in WASP activation by coincident GTPase and kinase signals. 相似文献
2.
Cdc42-interacting protein 4 mediates binding of the Wiskott-Aldrich syndrome protein to microtubules
The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy. Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motility. The Wiskott-Aldrich syndrome protein (WASP) is a multi-domain protein involved in cytoskeletal organization. In a two-hybrid screen, we identified the protein Cdc42-interacting protein 4 (CIP4) as a WASP interactor. CIP4, like WASP, is a Cdc42 effector protein involved in cytoskeletal organization. We found that the WASP-CIP4 interaction is mediated by the binding of the Src homology 3 domain of CIP4 to the proline-rich segment of WASP. Cdc42 was not required for this interaction. Co-expression of CIP4 and green fluorescent protein-WASP in COS-7 cells led to the association of WASP with microtubules. In vitro experiments showed that CIP4 binds to microtubules via its NH(2) terminus. The region of CIP4 responsible for binding to active Cdc42 was localized to amino acids 383-417, and the mutation I398S abrogated binding. Deletion of the Cdc42-binding domain of CIP4 did not affect the colocalization of WASP with microtubules in vivo. We conclude that CIP4 can mediate the association of WASP with microtubules. This may facilitate transport of WASP to sites of substrate adhesion in hematopoietic cells. 相似文献
3.
Rohatgi R Nollau P Ho HY Kirschner MW Mayer BJ 《The Journal of biological chemistry》2001,276(28):26448-26452
The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites. 相似文献
4.
M Kato H Miki K Imai S Nonoyama T Suzuki C Sasakawa T Takenawa 《The Journal of biological chemistry》1999,274(38):27225-27230
WASP (Wiskott-Aldrich syndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP. First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton. In addition, we found that the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering. Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site. 相似文献
5.
Fukuoka M Suetsugu S Miki H Fukami K Endo T Takenawa T 《The Journal of cell biology》2001,152(3):471-482
We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation. 相似文献
6.
O'Sullivan E Kinnon C Brickell P 《The international journal of biochemistry & cell biology》1999,31(3-4):383-387
Wiskott-Aldrich Syndrome protein (WASP) is the product of the gene mutated in children with Wiskott-Aldrich Syndrome (WAS). It is a predominantly cytoplasmic protein, expressed only in haematopoietic cells. It binds in vivo to the adaptor proteins Nck and Grb2, to the cytoplasmic protein-tyrosine kinase Fyn and to the small Rho-like GTPase Cdc42, which is required for formation of filopodia in fibroblasts and macrophages. WASP also interacts, directly or indirectly, with the actin cytoskeleton. Together with studies of a closely related, ubiquitously expressed protein named N-WASP, these findings suggest that WASP is a component of signalling pathways that control reorganisation of the actin cytoskeleton in haematopoietic cells in response to external stimuli. In support of this idea, haematopoietic cells from WAS patients show defects in cytoskeletal organisation that compromise their ability to polarise and to migrate in response to physiological stimuli. These defects could account for many of the clinical features of WAS. WAS is now a candidate for gene therapy based on the delivery of a wild-type WASP gene to autologous haematopoietic stem cells. In addition, recent studies of cell defects in WAS patients suggest that it may prove possible, in time, to rescue WAS cells using more conventional drug therapies. 相似文献
7.
Cell migration plays key roles in physiological and pathological phenomena, such as development and oncogenesis. The adaptor proteins Grb2, CrkII, and Nck1 are composed of only a single Src homology 2 domain and some Src homology 3 domains, giving specificity to each signal transduction pathway. However, little is known about the relationships between their adaptor proteins and cell migration, which are regulated by the G protein-coupled receptor. Here we showed that Nck1, but not Grb2 or CrkII, mediated the inhibition of cell migration induced by the endothelin-1 and endothelin A receptor. The small interference RNA and dominant negative mutants of Nck1 diminished the endothelin-1-induced inhibition of cell migration. Although overexpression of wild-type Nck1 was detected in the cytosol and did not affect cell migration, expression of the myristoylation signal sequence-conjugated Nck1 was detected in the membrane and induced activation of Cdc42 and c-Jun N-terminal kinase, inhibiting cell migration. Taken together, these results suggest that the endothelin A receptor transduces the signal of inhibition of cell migration through Cdc42-dependent c-Jun N-terminal kinase activation by using Nck1. 相似文献
8.
SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, betaPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that betaPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The betaPIX.WASP.SPIN90.Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90.betaPIX.WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90.Nck and SPIN90.betaPIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90.betaPIX.WASP complex and Nck. It thus appears that the interaction of the betaPIX.WASP.SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation. 相似文献
9.
The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible. 相似文献
10.
Phosphatidylinositol 4,5-biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck,WIP, and Grb2 总被引:10,自引:0,他引:10
Benesch S Lommel S Steffen A Stradal TE Scaplehorn N Way M Wehland J Rottner K 《The Journal of biological chemistry》2002,277(40):37771-37776
Wiskott-Aldrich syndrome protein (WASP)/Scar family proteins promote actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. While Scar/WAVE proteins are thought to be involved in lamellipodia protrusion, the hematopoietic WASP has been implicated in various actin-based processes such as chemotaxis, podosome formation, and phagocytosis. Here we show that the ubiquitously expressed N-WASP is essential for actin assembly at the surface of endomembranes induced as a consequence of increased phosphatidylinositol 4,5-biphosphate (PIP2) levels. This process resulting in the motility of intracellular vesicles at the tips of actin comets involved the recruitment of the Src homology 3 (SH3)-SH2 adaptor proteins Nck and Grb2 as well as of WASP interacting protein (WIP). Reconstitution of vesicle movement in N-WASP-defective cells by expression of various N-WASP mutant proteins revealed three independent domains capable of interaction with the vesicle surface, of which both the WH1 and the polyproline domains contributed significantly to N-WASP recruitment and/or activation. In contrast, the direct interaction of N-WASP with the Rho-GTPase Cdc42 was not required for reconstitution of vesicle motility. Our data reveal a distinct cellular phenotype for N-WASP loss of function, which adds to accumulating evidence that the proposed link between actin and membrane dynamics may, at least partially, be reflected by the actin-based movement of vesicles through the cytoplasm. 相似文献
11.
Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP 总被引:6,自引:0,他引:6
Labno CM Lewis CM You D Leung DW Takesono A Kamberos N Seth A Finkelstein LD Rosen MK Schwartzberg PL Burkhardt JK 《Current biology : CB》2003,13(18):1619-1624
Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse. 相似文献
12.
Treatment of cells with epidermal growth factor (EGF) promotes the activation of the small GTP-binding protein Cdc42, as well as its phosphorylation in cells. The EGF-dependent phosphorylation of Cdc42 occurs at tyrosine 64 in the Switch II domain and appears to be mediated through the Src tyrosine kinase, because both the expression of a dominant-negative Src mutant (mouse Src(K297R)) and treatment of cells with the Src kinase inhibitor PP2 blocks the EGF-stimulated phosphorylation of Cdc42, whereas expression of an activated Src mutant (Src(Y529F)) promotes phosphorylation in the absence of EGF treatment. The EGF-stimulated phosphorylation of Cdc42 is not required for its activation, nor does it directly affect the interactions of activated Cdc42 with target/effector proteins including PAK, ACK, WASP, or IQGAP. However, the EGF-stimulated phosphorylation of Cdc42 is accompanied by an enhancement in the interaction of Cdc42 with the Rho-GDP dissociation inhibitor (RhoGDI). The EGF-stimulated activation of Cdc42 does require activated Src, as well as the Vav2 protein, a member of the Dbl family of guanine nucleotide exchange factors. Src catalyzes the tyrosine phosphorylation of Vav2, and overexpression of Vav2 together with activated Src (Src(Y529F)) can completely bypass the need for EGF to promote the activation of Cdc42. Thus, EGF signaling through Src appears to have dual regulatory effects on Cdc42: 1). it leads to the activation of Cdc42 as mediated by the Vav2 guanine nucleotide exchange factor, and 2). it results in the phosphorylation of Cdc42, which stimulates the binding of RhoGDI, perhaps to direct the movement of Cdc42 to a specific cellular site to trigger a signaling response, because Cdc42-RhoGDI interactions are essential for Cdc42-induced cellular transformation. 相似文献
13.
Rich, a rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1. 总被引:9,自引:0,他引:9
A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1. 相似文献
14.
Tomasevic N Jia Z Russell A Fujii T Hartman JJ Clancy S Wang M Beraud C Wood KW Sakowicz R 《Biochemistry》2007,46(11):3494-3502
The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level. 相似文献
15.
Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence. 相似文献
16.
Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains. 总被引:18,自引:4,他引:14
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
O M Rivero-Lezcano A Marcilla J H Sameshima K C Robbins 《Molecular and cellular biology》1995,15(10):5725-5731
In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, we report the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain. 相似文献
17.
Joanna R. Watson Helen M. Fox Daniel Nietlispach Jennifer L. Gallop Darerca Owen Helen R. Mott 《The Journal of biological chemistry》2016,291(26):13875-13890
Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. 相似文献
18.
Marcus Lettau Jennifer Pieper Alyn Gerneth Beate Lengl‐Janßen Matthias Voss Andreas Linkermann Hendrik Schmidt Christoph Gelhaus Matthias Leippe Dieter Kabelitz Ottmar Janssen 《Protein science : a publication of the Protein Society》2010,19(4):658-669
Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes. 相似文献
19.
The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules. 相似文献
20.
Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells. 总被引:6,自引:1,他引:5
下载免费PDF全文
![点击此处可从《Molecular biology of the cell》网站下载免费的PDF全文](/ch/ext_images/free.gif)
H Y She S Rockow J Tang R Nishimura E Y Skolnik M Chen B Margolis W Li 《Molecular biology of the cell》1997,8(9):1709-1721
Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton. 相似文献