The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.     

Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.     

Interaction of guanylyl cyclase C with SH3 domain of Src tyrosine kinase. Yet another mechanism for desensitization

Protein-protein interactions mediated by the Src homology 3 (SH3) domain have been implicated in the regulation of receptor functions for subcellular localization of proteins and the reorganization of cytoskeleton. The experiments described in this article begin to identify the interaction of the SH3 domain of Src tyrosine kinase with the guanylyl cyclase C receptor after activation with Escherichia coli heat-stable enterotoxin (ST). Only one of two post-translationally modified forms of guanylyl cyclase C from T84 colonic carcinoma cells bind to GST-SH3 fusion protein of Src and Hck tyrosine kinases. Interestingly, the GST-Src-SH3 fusion protein showed 2-fold more affinity to native guanylyl cyclase C in solution than the GST-Hck-SH3 fusion protein. The affinity of the GST-Src-SH3 fusion protein to guanylyl cyclase C increased on desensitization of receptor in vivo. An in vitro cyclase assay in the presence of GST-Src-SH3 fusion protein indicated inhibition of the catalytic activity of guanylyl cyclase C. The catalytic domain recombinant protein (GST-GCD) of guanylyl cyclase C could pull-down a 60-kDa protein that reacted with Src tyrosine antibody and also showed autophosphorylation. These data suggest that SH3 domain-mediated protein-protein interaction with the catalytic domain of guanylyl cyclase C inhibited the cyclase activity and that such an interaction, possibly mediated by Src tyrosine kinase or additional proteins, might be pivotal for the desensitization phenomenon of the guanylyl cyclase C receptor.     

A modified immunoblot method to identify substrates of protein kinases

While protein kinases are key components in multiple cellular processes, efficient identification of cognate in vivo substrates remains challenging. Here we describe a powerful method to screen potential substrates of protein kinases by partial transfer of proteins from a 2D-PAGE gel to a Western blot membrane. This approach allowed precise pinpointing of candidate substrate spots in the 2D gel, and identifying physiological substrates of protein kinases in Mycobacterium tuberculosis.     

Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, C-terminal Src kinase (Csk)

Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.     

Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase.

The tyrosine kinase activity intrinsic to the insulin receptor is thought to be important in eliciting the intracellular responses to insulin; however, it has been difficult to determine the biochemical functions of the proteins which are substrates for this receptor. Treatment of Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor (CHO.T) with insulin results in a 38 +/- 11 (mean +/- S.E., n = 9)-fold increase in a phosphatidylinositol (PtdIns) kinase activity in anti-phosphotyrosine immunoprecipitates of whole cell lysates. One minute of treatment of cells with insulin causes a dramatic increase in the PtdIns kinase activity in the anti-phosphotyrosine immunoprecipitates; the activity peaks within 5 min and remains elevated for at least 60 min after addition of insulin to the cells. This response is only slightly delayed compared with the time course we observe for activation of the insulin receptor tyrosine kinase. The insulin dose-response curves are also very similar for the activation of the insulin receptor tyrosine kinase activity and for the appearance of PtdIns kinase in the anti-phosphotyrosine immunoprecipitates. Stimulation of the endogenous insulin receptor of CHO cells also results in the association of PtdIns kinase activity with phosphotyrosine-containing proteins. However, CHO cells are less sensitive to insulin than CHO.T cells, and the maximal PtdIns kinase activity in antiphosphotyrosine immunoprecipitates from CHO cells is one-sixth that of CHO.T cells. In contrast, immunoprecipitates from CHO.T cells made with anti-insulin receptor antibodies do not contain significant levels of PtdIns kinase activity. This demonstrates that the PtdIns kinase is either a substrate for the insulin receptor tyrosine kinase or is tightly associated with another tyrosine phosphoprotein, which is not the insulin receptor.     

Structural basis for domain-domain communication in a protein tyrosine kinase, the C-terminal Src kinase

The catalytic activity of protein tyrosine kinases is commonly regulated by domain-domain interactions. The C-terminal Src kinase (Csk) contains a catalytic domain and the regulatory SH3 and SH2 domains. Both the presence of the regulatory domains and binding of specific phosphotyrosine-containing proteins to the SH2 domain activate Csk. The structural basis for both modes of activation is investigated here. First, the SH3-SH2 linker is crucial for Csk activation. Mutagenic and kinetic studies demonstrate that this activation is mediated by a cation-pi interaction between Arg68 and Trp188. Second, Ala scanning and kinetic analyses on residues in the SH2-catalytic domain interface identify three functionally distinct types of residues in mediating the communication between the SH2 and the catalytic domains. Type I residues are important in mediating a ligand-triggered activation of Csk because their mutation severely reduces Csk activation by the SH2 domain ligand. Type II residues are involved in suppressing Csk activity, and their mutation activates Csk, but makes Csk less sensitive to activation by the SH2 ligand. Both type I and type II residues are likely involved in mediating SH2 ligand-triggered activation of Csk. Type III residues are those located in the SH2 domain whose mutation severely decreases Csk catalytic activity without affecting the SH2 ligand-triggered activation. These residues likely mediate SH2 activation of Csk regardless of SH2-ligand interaction. These studies lead us to propose a domain-domain communication model that provides functional insights into the topology of Csk family of protein tyrosine kinases.     

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 regulates the migration of Langerhans cells from the epidermis to draining lymph nodes

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-alpha-induced migration of LCs. The emigration of dendritic cells expressing I-A(b+) from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3beta was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration.     

A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

Electroporation is an important approach for genetic engineering experiments allowing for introduction of foreign DNA in a selected host. Here, we describe for the first time the use of glycine betaine as an osmoprotectant for electroporation of gram-positive bacteria Bacillus subtilis. High electroporation efficiency (up to 5×10(5) cfu/μg) was obtained using 7.5% glycine betaine. The new method improved the transformation efficiency of B. subtilis with linear integrative DNA nearly 700-fold compared with existing Bacillus transformation techniques.     

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol- anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.     

Fibroblast growth factor receptor-1-mediated endothelial cell proliferation is dependent on the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-alpha/FGFR-1 (alphaR/FR) wild type and mutant Y463F receptors. The kinase activity of alphaR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant alphaR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type alphaR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.     

A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain

This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXXS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.     

c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein.

The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase. Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.     

Protein kinase C activation inhibits tyrosine phosphorylation of Cbl and its recruitment of Src homology 2 domain-containing proteins.

One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCalpha and PKCtheta can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKCalpha and PKCtheta in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.     

Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes.

Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.