The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

Src protein-tyrosine kinase structure and regulation

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPalpha displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the alphaD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu(4)Tyr).     

Structural basis for the recognition of c-Src by its inactivator Csk

The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 A resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.     

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.     

Src-dependent,neutrophil-mediated vascular hyperpermeability and beta-catenin modification

The hyperpermeability response of microvessels in inflammation involves complex signaling reactions and structural modifications in the endothelium. Our goal was to determine the role of Src-family kinases (Src) in neutrophil-mediated venular hyperpermeability and possible interactions between Src and endothelial barrier components. We found that inhibition of Src abolished the increases in albumin permeability caused by C5a-activated neutrophils in intact, perfused coronary venules, as well as in cultured endothelial monolayers. Activated neutrophils increased Src phosphorylation at Tyr416, which is located in the catalytic domain, and decreased phosphorylation at Tyr527 near the carboxyl terminus, events consistent with reports that phosphorylating and transforming activities of Src are upregulated by Tyr416 phosphorylation and negatively regulated by Tyr527 phosphorylation. Furthermore, neutrophil stimulation resulted in association of Src with the endothelial junction protein beta-catenin and beta-catenin tyrosine phosphorylation. These phenomena were abolished by blockage of Src activity. Taken together, our studies link for the first time neutrophil-induced hyperpermeability to a pathway involving Src kinase activation, Src/beta-catenin association, and beta-catenin tyrosine phosphorylation in the microvascular endothelium.     

Chemical rescue of a mutant protein-tyrosine kinase

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.     

Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function.

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein-serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34cdc2 is phosphorylated on two sites, one of which has been identified as Tyr15. Dephosphorylation of Tyr15 regulates the initiation of mitosis. To understand more completely the regulation of p34cdc2 kinase activity, we have identified the second site of phosphorylation as Thr167, a residue conserved amongst all p34cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thr167 phosphorylation is required for p34cdc2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thr167 might also play a role in the exit from mitosis.     

Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine

Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl₂) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg²⁺ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl₂ at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl₂-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.     

Src tyrosine kinase-dependent migratory effects of antithrombin in leukocytes

Tyrosine kinases are known to play a critical role in the regulation of leukocyte function. Antithrombin mediates its effects via syndecan-4 which is known to be linked to the Src tyrosine kinases. In this study, we investigated the role of Src tyrosine kinases in antithrombin-regulated leukocyte migration and Src tyrosine kinase phosphorylation in response to stimulation with antithrombin. Neutrophils and monocytes obtained from forearm venous blood were pre-treated by various Src-family selective tyrosine kinase inhibitors with or without antithrombin followed by washing and assessment of their migratory response toward antithrombin, interleukin-8, or RANTES using Boyden microchemotaxis chambers. Activation status of the two major Src tyrosine kinase phosphorylation sides Tyr416 and Tyr527 was tested using Western blot analysis. Dose-dependent reversal of the antithrombin-mediated effects on neutrophil and monocyte migration was induced by the selective Src kinase inhibitors PP1 and PP2. In Western blot analyses, antithrombin increased Tyr416 and decreased Tyr527 phosphorylation of Src tyrosine kinases in a time- and dose-dependent manner. Moreover, co-incubation with antithrombin lowered the level of RANTES-induced Tyr416 phosphorylation. Therefore, Src tyrosine kinases linked to signaling of antithrombin-binding sites on leukocytes may play an important role in modulating effects on cells function.     

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.     

Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells

We have shown previously that the Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. The importance of Lyn to red cell maturation has been highlighted by Lyn-/- mice developing anemia. Here we show that Lyn interacts with C-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators C-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk). Lyn phosphorylated Cbp on several tyrosine residues, including Tyr314, which recruited Csk/Ctk to suppress Lyn kinase activity. Intriguingly, phosphorylated Tyr314 also bound suppressor of cytokine signaling 1 (SOCS1), another well characterized negative regulator of cell signaling, resulting in elevated ubiquitination, and degradation of Lyn. In Epo-responsive primary cells and cell lines, Lyn rapidly phosphorylated Cbp, suppressing Lyn kinase activity via Csk/Ctk within minutes of Epo stimulation; hours later, SOCS1 bound to Cbp and was involved in the ubiquitination and turnover of Lyn protein. Thus, a single phosphotyrosine residue on Cbp coordinates a two-phase process involving distinct negative regulatory pathways to inactivate, then degrade, Lyn.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Regulation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-5 in vascular smooth muscle cells involves the phosphatase Shp2

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by approximately 35%. Physiologic SMC GRK5 activity also increased PDGFRbeta association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRbeta Tyr(1009) (the docking site for Shp2), and reduced phosphorylation of PDGFRbeta Tyr(1021). Consistent with having increased PDGFRbeta-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRbeta inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRbeta/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRbeta/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRbetas. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRbeta in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRbeta inositol phosphate signaling and enhances PDGFRbeta-triggered Src activation.     

Regulation of c-Src activity by the expression of wild-type v-Src and its kinase-dead double Y416F-K295N mutant

Active, wild-type v-Src and its kinase-dead double Y416F-K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F-K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.     

Positive and negative regulation of type II TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.

The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively.     

Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation

CD45 is a transmembrane, two-domain protein-tyrosine phosphatase expressed exclusively in nucleated hematopoietic cells. The Src family kinase, Lck, is a major CD45 substrate in T cells and CD45 dephosphorylation of Lck is important for both T cell development and activation. However, how the substrate specificity of phosphatases such as CD45 is achieved is not well understood. Analysis of the interaction between the cytoplasmic domain of CD45 and its substrate, Lck, revealed that the active, membrane-proximal phosphatase domain of CD45 (CD45-D1) bound to the phosphorylated Lck kinase domain, the SH2 domain, and the unique N-terminal region of Lck. The second, inactive phosphatase domain (CD45-D2) bound only to the kinase domain of Lck. CD45-D2 was unable to bind phosphotyrosine, and its interaction with the kinase domain of Lck was independent of tyrosine phosphorylation. The binding of CD45-D2 was localized to subdomain X (SD10) of Lck. CD45-D2 bound similarly to Src family kinases but bound Csk to a lesser extent and did not bind significantly to the less related kinase, Erk1. CD45 dephosphorylated Lck and Src at similar rates but dephosphorylated Csk and Erk1 at lower rates. Replacement of Erk1 SD10 with that of Lck resulted in the binding of CD45-D2 and the conversion of Erk1 to a more efficient CD45 substrate. This demonstrates a role for CD45-D2 in binding substrate and identifies the SD10 region in Lck as a novel site involved in substrate recognition.