ArgBP2gamma interacts with Akt and p21-activated kinase-1 and promotes cell survival

Akt/protein kinase B is a major cell survival pathway through phosphorylation of proapoptotic proteins Bad and Bax and of additional apoptotic pathways linked to Forkhead proteins glycogen synthase kinase-3beta and ASK1. To further explore the mechanism by which Akt regulates cell survival, we identified an Akt interaction protein by yeast two-hybrid screening. It is highly homologous to ARG-binding protein 2 (ArgBP2) with splicing exon 8 of the coding region of the ArgBP2. As two splicing isoforms (ArgBP2alpha and -beta) of ArgBP2 have been identified (Wang, B., Golemis, E. A., and Kruh, G. D. (1997) J. Biol. Chem. 272, 17542-17550), it was named ArgBP2gamma. ArgBP2gamma contains four Akt phosphorylation consensus sites, a SoHo motif, and three Src homology (SH) 3 domains and binds to C-terminal proline-rich motifs of Akt through its first and second SH3 domains. It also interacts with p21-activated protein kinase (PAK1) via its first and third SH3 domains, indicating the SH3 domains of ArgBP2gamma as docking sites for Akt and PAK1. Akt phosphorylates ArgBP2gamma in vitro and in vivo. Expression of ArgBP2gamma induces PAK1 activity and overrides apoptosis induced by ectopic expression of Bad or DNA damage. Nonphosphorylatable ArgBP2gamma-4A and SH3 domain-truncated mutant ArgBP2gamma inhibit Akt-induced PAK1 activation and reduce Akt and PAK1 phosphorylation of Bad and antiapoptotic function. These data indicate that ArgBP2gamma is a physiological substrate of Akt, functions as an adaptor for Akt and PAK1, and plays a role in Akt/PAK1 cell survival pathway.     

Molecular Dynamics Simulations Reveal that Tyr-317 Phosphorylation Reduces Shc Binding Affinity for Phosphotyrosyl Residues of Epidermal Growth Factor Receptor

The Src homology 2 (SH2) and collagen domain protein Shc plays a pivotal role in signaling via tyrosine kinase receptors, including epidermal growth factor receptor (EGFR). Shc binding to phospho-tyrosine residues on activated receptors is mediated by the SH2 and phospho-tyrosine binding (PTB) domains. Subsequent phosphorylation on Tyr-317 within the Shc linker region induces Shc interactions with Grb2-Son of Sevenless that initiate Ras-mitogen-activated protein kinase signaling. We use molecular dynamics simulations of full-length Shc to examine how Tyr-317 phosphorylation controls Shc conformation and interactions with EGFR. Our simulations reveal that Shc tyrosine phosphorylation results in a significant rearrangement of the relative position of its domains, suggesting a key conformational change. Importantly, computational estimations of binding affinities show that EGFR-derived phosphotyrosyl peptides bind with significantly more strength to unphosphorylated than to phosphorylated Shc. Our results unveil what we believe is a novel structural phenomenon, i.e., tyrosine phosphorylation of Shc within its linker region regulates the binding affinity of SH2 and PTB domains for phosphorylated Shc partners, with important implications for signaling dynamics.     

Involvement of palladin and alpha-actinin in targeting of the Abl/Arg kinase adaptor ArgBP2 to the actin cytoskeleton

Palladin and alpha-actinin are major components of stress fiber dense bodies, cardiomyocyte Z-discs and neuronal synapses. They function as structural molecules and cytoskeletal regulators but also as docking sites to other proteins. Both antisense and transient overexpression experiments have shown that palladin plays an important role in the regulation of actin cytoskeleton. ArgBP2 is a multi-domain scaffolding protein which shares both the tissue distribution and subcellular localization with palladin. ArgBP2 is directly linked to intracellular signaling cascades by its interaction with Abl family kinases, Pyk2 and the ubiquitin ligase Cbl. It has several actin associated binding partners and has been shown to regulate cytoskeletal dynamics. Here, we show by in vivo and in vitro methods that palladin's amino-terminal poly-proline sequences directly interact with the first carboxy-terminal SH3 domain of ArgBP2. We further demonstrate a direct interaction between alpha-actinin and the amino-terminal segment of ArgBP2. Immunoprecipitation and targeting assays suggest that a three-way complex of the proteins occurs in vivo. The interactions provide an explanation to the previously observed Z-disc-specific localization of ArgBP2 and indicate interplay between signaling adaptors and structural proteins of the Z-disc.     

ArgBP2 and the SoHo family of adapter proteins in oncogenic diseases

ArgBP2, a member of the SoHo family of adapter proteins, is a regulator of actin-dependent processes such as cell adhesion and migration. Recent data from our lab revealed that by regulating adhesion and migration of pancreatic cancer cells, ArgBP2 is endowed with an anti-tumoral function. We could show that part of the molecular mechanism involved the interaction of ArgBP2 with the Arp2/3 activator WAVE1, the tyrosine phosphatase PTP-PEST, and the tyrosine kinase c-Abl. As ArgBP2 shares common structural organization and overlapping functions with the two other members of this protein family, CAP and Vinexin, it raises the question whether these two other proteins could also be involved in cancer diseases. The control of cell migration being an important issue in tumor treatment, these recent findings suggest that ArgBP2 family-dependent signaling pathways represents potential targets for the development of therapeutic strategies, and highlight the importance of elucidating their molecular mechanisms of cytoskeletal regulation.Key words: ArgBP2, pancreatic cancer, SoHo family of proteins, adapter protein, CAP/ponsin, vinexin, cell signaling, actin cytoskeleton, adhesion, migrationAcquisition of migration properties by cancerous cells is a crucial feature for cancer cell invasion and metastasis and, therefore, the control of cell migration is an important issue in tumor treatment.¹ As molecular mechanisms governing cell motility are common to non-neoplasic cells and cancer cells, the elucidation of these mechanisms is crucial in order to better understand how the loss of control of cell adhesion, migration and cytoskeletal organization participate to tumorigenesis.     

ArgBP2 and the SoHo family of adapter proteins in oncogenic diseases

ArgBP2, a member of the SoHo family of adapter proteins, is a regulator of actin-dependent processes such as cell adhesion and migration. Recent data from our lab revealed that by regulating adhesion and migration of pancreatic cancer cells, ArgBP2 is endowed with an anti-tumoral function. We could show that part of the molecular mechanism involved the interaction of ArgBP2 with the Arp2/3 activator WAVE1, the tyrosine phosphatase PTP-PEST, and the tyrosine kinase c-Abl. As ArgBP2 shares common structural organization and overlapping functions with the two other members of this protein family, CAP and Vinexin, it raises the question whether these two other proteins could also be involved in cancer diseases. The control of cell migration being an important issue in tumor treatment, these recent findings suggest that ArgBP2 family-dependent signaling pathways represents potential targets for the development of therapeutic strategies, and highlight the importance of elucidating their molecular mechanisms of cytoskeletal regulation.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

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.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

Src phosphorylation of Alix/AIP1 modulates its interaction with binding partners and antagonizes its activities

Alix/AIP1 is an adaptor protein involved in regulating the function of receptor and cytoskeleton-associated tyrosine kinases. Here, we investigated its interaction with and regulation by Src. Tyr319 of Alix bound the isolated Src homology-2 (SH2) domain and was necessary for interaction with intact Src. A proline-rich region in the C terminus of Alix bound the Src SH3 domain, but this interaction was dependent on the release of the Src SH2 domain from its Src internal ligand either by interaction with Alix Tyr319 or by mutation of Src Tyr527. Src phosphorylated Alix at a C-terminal region rich in tyrosines, an activity that was stimulated by the presence of the Alix binding partner SETA/CIN85. Phosphorylation of Alix by Src caused it to translocate from the membrane and cytoskeleton to the cytoplasm and reduced its interaction with binding partners SETA/CIN85, epidermal growth factor receptor, and Pyk2. As a consequence of this, Src antagonized the negative regulation of receptor tyrosine kinase internalization and cell adhesion by Alix. We propose a model whereby Src antagonizes the effects of Alix by phosphorylation of its C terminus, leading to the disruption of interactions with target proteins.     

Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control

Cortactin is an important molecular scaffold for actin assembly and organization. Novel mechanistic functions of cortactin have emerged with more interacting partners identified, revealing its multifaceted roles in regulating actin cytoskeletal networks that are necessary for endocytosis, cell migration and invasion, adhesion, synaptic organization and cell morphogenesis. These processes are mediated by its multi-domains binding to F-actin and Arp2/3 complex and various SH3 targets. Furthermore, its role in actin remodeling is subjected to regulation by tyrosine and serine/threonine kinases. Elucidating the mechanisms underlying cortactin phosphorylation and its functional consequences would provide new insights to various aspects of cell dynamics control.     

Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.     

Regulation of SHP-1 tyrosine phosphatase in human platelets by serine phosphorylation at its C terminus

SHP-1 is a Src homology 2 (SH2) domain-containing tyrosine phosphatase that plays an essential role in negative regulation of immune cell activity. We describe here a new model for regulation of SHP-1 involving phosphorylation of its C-terminal Ser591 by associated protein kinase Calpha. In human platelets, SHP-1 was found to constitutively associate with its substrate Vav1 and, through its SH2 domains, with protein kinase Calpha. Upon activation of either PAR1 or PAR4 thrombin receptors, the association between the three proteins was retained, and Vav1 became phosphorylated on tyrosine and SHP-1 became phosphorylated on Ser591. Phosphorylation of SHP-1 was mediated by protein kinase C and negatively regulated the activity of SHP-1 as demonstrated by a decrease in the in vitro ability of SHP-1 to dephosphorylate Vav1 on tyrosine. Protein kinase Calpha therefore critically and negatively regulates SHP-1 function, forming part of a mechanism to retain SHP-1 in a basal active state through interaction with its SH2 domains, and phosphorylating its C-terminal Ser591 upon cellular activation leading to inhibition of SHP-1 activity and an increase in the tyrosine phosphorylation status of its substrates.     

Adapter protein SH2B1beta binds filamin A to regulate prolactin-dependent cytoskeletal reorganization and cell motility

Prolactin (PRL) regulates cytoskeletal rearrangement and cell motility. PRL-activated Janus tyrosine kinase 2 (JAK2) phosphorylates the p21-activated serine-threonine kinase (PAK)1 and the Src homology 2 (SH2) domain-containing adapter protein SH2B1β. SH2B1β is an actin-binding protein that cross-links actin filaments, whereas PAK1 regulates the actin cytoskeleton by different mechanisms, including direct phosphorylation of the actin-binding protein filamin A (FLNa). Here, we have used a FLNa-deficient human melanoma cell line (M2) and its derivative line (A7) that stably expresses FLNa to demonstrate that SH2B1β and FLNa are required for maximal PRL-dependent cell ruffling. We have found that in addition to two actin-binding domains, SH2B1β has a FLNa-binding domain (amino acids 200-260) that binds directly to repeats 17-23 of FLNa. The SH2B1β-FLNa interaction participates in PRL-dependent actin rearrangement. We also show that phosphorylation of the three tyrosines of PAK1 by JAK2, as well as the presence of FLNa, play a role in PRL-dependent cell ruffling. Finally, we show that the actin- and FLNa-binding-deficient mutant of SH2B1β (SH2B1β 3Δ) abolished PRL-dependent ruffling and PRL-dependent cell migration when expressed along with PAK1 Y3F (JAK2 tyrosyl-phosphorylation-deficient mutant). Together, these data provide insight into a novel mechanism of PRL-stimulated regulation of the actin cytoskeleton and cell motility via JAK2 signaling through FLNa, PAK1, and SH2B1β. We propose a model for PRL-dependent regulation of the actin cytoskeleton that integrates our findings with previous studies.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.     

SHP-2 inhibits tyrosine phosphorylation of Cas-L and regulates cell migration

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase, SHP-2, plays an important role in cell migration by interacting with various proteins. In this report, we demonstrated that SHP-2 inhibits tyrosine phosphorylation of Crk-associated substrate lymphocyte type (Cas-L), a docking protein which mediates cell migration, and found that SHP-2 negatively regulates migration of A549 lung adenocarcinoma cells induced by fibronectin (FN). We showed that overexpressed SHP-2 co-localizes with Cas-L at focal adhesions and that exogenous expression of SHP-2 abrogates cell migration mediated by Cas-L. SHP-2 inhibits tyrosine phosphorylation of Cas-L, and associates with Cas-L to form a complex in a tyrosine phosphorylation-dependent manner. Finally, immunoprecipitation experiments with deletion mutants revealed that both SH2 domains of SHP-2 are necessary for this association. These results suggest that SHP-2 regulates tyrosine phosphorylation of Cas-L, hence opposing the effect of kinases, and SHP-2 is a negative regulator of cell migration mediated by Cas-L.     

The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.     

Src redox regulation: there is more than meets the eye

Src-family kinases are critically involved in the control of cytoskeleton organization and in the generation of integrin-dependent signaling responses, inducing tyrosine phosphorylation of many signaling and cytoskeletal proteins. Activity of the Src family of tyrosine kinases is tightly controlled by inhibitory phosphorylation of a carboxy-terminal tyrosine residue, inducing an inactive conformation through binding with its SH2 domain. Dephosphorylation of C-ter tyrosine, as well as its deletion of substitution with phenylalanine in oncogenic Src kinases, leads to autophosphorylation at a tyrosine in the activation loop, thereby leading to enhanced Src activity. Beside this phophorylation/dephosphorylation circuitry, cysteine oxidation has been recently reported as a further mechanism of enzyme activation. Mounting evidence describes Src activation via its redox regulation as a key outcome in several circumstances, including growth factor and cytokines signaling, integrin-mediated cell adhesion and motility, membrane receptor cross-talk as well in cell transformation and tumor progression. Among the plethora of data involving Src kinase in physiological and pathophysiological processes, this review will give emphasis to the redox component of the regulation of this master kinase.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.     

The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells

The mechanisms leading to focal adhesion kinase (FAK) activation remain obscure. We have investigated the role of the adaptor protein Shb in cell spreading and the regulation of FAK phosphorylation in immortalised brain endothelial (IBE) cells. Fibroblast growth factor 2 (FGF-2) stimulation lead to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding (PTB) domain of Shb. IBE cells overexpressing wild-type or R522K Shb (with an inactive Src homology 2 (SH2) domain) displayed increased FAK phosphorylation as well as enhanced spreading when seeded on collagen. FGF-2-induced tyrosine phosphorylation of Shb was dependent upon Src activity but independent of FAK activation. The use of Rat-1 fibroblasts overexpressing a temperature sensitive v-Src (tsLA29) confirmed that active Src enhanced Shb phosphorylation. The data indicate that Shb binds directly to FAK and regulates its phosphorylation leading to enhanced cell spreading in a Src-dependent manner.