Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.     

Profiling the global tyrosine phosphorylation state

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome.     

Identification of CrkL-SH3 binding proteins from embryonic murine brain: implications for Reelin signaling during brain development

The Crk and Crk-like (CrkL) adaptor proteins play important roles in numerous signaling pathways, bridging tyrosine kinase substrates to downstream signaling effectors by virtue of their phosphotyrosine-binding SH2 domains and their effector-binding SH3 domains. Critical to understanding the diverse roles of Crk/CrkL is the identification of tissue- and signal-specific tyrosine phosphorylated substrates to which they are recruited and the tissue-specific effector proteins they chaperone into signaling complexes. Crk and CrkL are known biochemically and genetically to be essential mediators of Reelin/Disabled-1 (Dab1) signaling, which governs proper mammalian brain development. Multimeric Reelin clusters its receptors as well as the receptor-bound intracellular scaffolding protein Dab1. Clustering induces Fyn/Src-dependent Dab1 tyrosine phosphorylation, which recruits Crk/CrkL and SH3-bound effectors. Previously, 21 Crk/CrkL-SH3 binding proteins were identified from diverse cell types. We present here the proteomic identification of 101 CrkL-SH3 binding proteins from embryonic murine brain. The identified proteins are enriched in the Crk/CrkL-SH3 binding motif and signaling activities regulating cell adhesion and motility. These results suggest Reelin-induced Dab1 tyrosine phosphorylation may generate a multifaceted signaling scaffold containing a rich array of Crk/CrkL-SH3 binding effectors and may explain a growing diversity of cellular activities suggested to be influenced by Reelin/Dab1 signaling.     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.     

Regulation of the SHP-2 tyrosine phosphatase by a novel cholesterol- and cell confluence-dependent mechanism

Endothelial cells approaching confluence exhibit marked decreases in tyrosine phosphorylation of receptor tyrosine kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close correlation in endothelial cells between membrane cholesterol and tyrosine phosphorylation of adherens junctions proteins. Here, we probe the mechanistic basis for this correlation. We find that as endothelial cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl phosphate, strongly suggesting that this binding was mediated by other regions of SHP-2 beside its SH2 domains. Annexin II, previously implicated in cholesterol trafficking, was associated in a complex with SHP-2, and both proteins localized to adhesion bands in confluent endothelial monolayers. These studies reveal a novel, cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.

Many cellular signaling proteins contain SH3 (Src homology 3) domains that mediate protein interactions via specific proline-containing peptides. Unlike SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 binding site, the regulatory mechanism for SH3 interactions is unclear. p120 RasGAP (GTPase-activating protein), which contains an SH3 domain flanked by two SH2 domains, forms an abundant SH2-mediated complex with p190 RhoGAP in cells expressing activated tyrosine kinases. We have identified two closely linked tyrosine-containing peptides in p190 that bind simultaneously to the RasGAP SH2 domains upon p190 phosphorylation. This interaction is expected to bring the two SH2 domains into close proximity. Consequently, RasGAP undergoes a conformational change that results in a 100-fold increase in the accessibility of the target binding surface of its SH3 domain. These results indicate that the tandem arrangement of SH2 and SH3 domains found in a variety of cellular signaling proteins can provide a conformational mechanism for regulating SH3-dependent interactions through tyrosine phosphorylation. In addition, it appears that the role of p190 in the RasGAP signaling complex is to promote additional protein interactions with RasGAP via its SH3 domain.     

Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains

Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.     

Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion

Activated neutrophils display an array of physiological responses, including initiation of the oxidative burst, phagocytosis, and cell migration, that are associated with cellular adhesion. Under conditions that lead to cellular adhesion, we observed rapid tyrosine phosphorylation of an intracellular protein with an approximate relative molecular mass of 92 kDa (p92). Phosphorylation of p92 was inducible when Mac-1 was activated by phorbol 12-myristate 13-acetate, the beta(2)-specific activating antibody CBR LFA-1/2, or interleukin-8 (77 amino acids). In addition, tyrosine phosphorylation of p92 was dependent on engagement of Mac-1 with ligand. Several observations suggest that this event may be an important step in the signaling pathway initiated by Mac-1 binding. p92 phosphorylation was specifically blocked with antibodies to CD11b, the alpha-subunit of Mac-1, and was rapidly reversible on disengagement of the integrin ligand interaction. Integrin-stimulated phosphorylation of p92 created binding sites that were recognized in vitro by the SH2 domains of c-CrkII and Src. Our observations suggest that neutrophil adhesion mediated through the binding of the beta(2)-integrin Mac-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interactions via SH2 domains, a key process shared with growth factor signaling pathways.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

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.     

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.     

Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling

Background

Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/Principal Findings

We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/Significance

This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.     

Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor.

src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.     

Dissecting the interaction of SHP-2 with PZR, an immunoglobulin family protein containing immunoreceptor tyrosine-based inhibitory motifs

Tyrosine phosphorylation of membrane proteins plays a crucial role in cell signaling by recruiting Src homology 2 (SH2) domain-containing signaling molecules. Recently, we have isolated a transmembrane protein designated PZR that specifically binds tyrosine phosphatase SHP-2, which has two SH2 domains (Zhao, Z. J., and Zhao, R. (1998) J. Biol. Chem. 273, 29367-29372). PZR belongs to the immunoglobulin superfamily. Its intracellular segment contains four putative sites of tyrosine phosphorylation. By site-specific mutagenesis, we found that the tyrosine 241 and 263 embedded in the consensus immunoreceptor tyrosine-based inhibitory motifs VIYAQL and VVYADI, respectively, accounted for the entire tyrosine phosphorylation of PZR. The interaction between PZR and SHP-2 requires involvement of both tyrosyl residues of the former and both SH2 domains of the latter, since its was disrupted by mutating a single tyrosyl residue or an SH2 domain. Overexpression of catalytically inactive but not active forms of SHP-2 bearing intact SH2 domains in cells caused hyperphosphorylation of PZR. In vitro, tyrosine-phosphorylated PZR was efficiently dephosphorylated by the full-length form of SHP-2 but not by its SH2 domain-truncated form. Together, the data indicate that PZR serves not only as a specific anchor protein of SHP-2 on the plasma membrane but also as a physiological substrate of the enzyme. The coexisting binding and dephosphorylation of PZR by SHP-2 may function to terminate signal transduction initiated by PZR and SHP-2 and to set a threshold for the signal transduction to be initiated.     

Identification of Fyn-binding proteins in MC/9 mast cells using mass spectrometry

Fyn is a Src kinase known to have an essential role in mast cell degranulation induced following aggregation of the high affinity IgE-receptor. Although Fyn possesses SH2 and SH3 protein binding domains, the molecules that interact with Fyn have not been characterized in mast cells. We thus analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathway. On mass spectrometric analysis of proteins binding to the SH2 and SH3 domains of Fyn, we identified six proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. Among these proteins, vimentin and pyruvate kinase have not been shown to bind to Fyn. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased; and this interaction was via binding to the SH2, but not the SH3, domain of Fyn. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine phosphorylation of intracellular proteins including NTAL and LAT. The observation that vimentin and pyruvate kinase bind to Fyn provides additional insight into Fyn-mediated signaling pathways, and suggests a critical role for Fyn in mast cell degranulation in interacting with both cytosolic and structural proteins.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.     

Kindlin-2 Tyrosine Phosphorylation and Interaction with Src Serve as a Regulatable Switch in the Integrin Outside-in Signaling Circuit

Integrin-mediated cell-extracellular matrix (ECM) adhesion is critical for control of intracellular signaling; however, the mechanisms underlying this “outside-in” signaling are incompletely understood. Here we show that depletion of kindlin-2 impairs integrin outside-in signaling. Kindlin-2 is tyrosine-phosphorylated upon cell-ECM adhesion. Furthermore, kindlin-2 binds Src in a cell-ECM adhesion-regulatable fashion. At the molecular level, the kindlin-2·Src interaction is mediated by the kindlin-2 F0 and the Src SH2 and SH3 domains. Src activation increases kindlin-2 tyrosine phosphorylation and the kindlin-2·Src interaction. Conversely, inhibition of Src reduces kindlin-2 tyrosine phosphorylation and diminishes the kindlin-2·Src interaction. Finally, disruption of the kindlin-2·Src interaction, unlike depletion of kindlin-2, impairs neither cell-ECM adhesion nor cell-ECM adhesion-induced focal adhesion kinase Tyr-397 phosphorylation. However, it markedly inhibits cell-ECM adhesion-induced paxillin tyrosine phosphorylation, cell migration, and proliferation. These results suggest that kindlin-2 tyrosine phosphorylation and interaction with Src serve as a regulatable switch downstream of focal adhesion kinase in the integrin outside-in signaling circuit, relaying signals from cell-ECM adhesion to paxillin that control cell migration and proliferation.     

Regulation of c-SRC activity and function by the adapter protein CAS

SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.     

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.