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1.
Src-family kinases (SFKs) control a variety of biological processes, from cell proliferation and differentiation to cytoskeletal rearrangements. Abnormal activation of SFKs has been implicated in a wide variety of cancers and is associated with metastatic behavior (Yeatman, 2004). SFKs are maintained in an inactive state by inhibitory phosphorylation of their C-terminal region by C-terminal Src kinase (Csk). We have identified Drosophila Ankyrin-repeat, SH3-domain, and Proline-rich-region containing Protein (dASPP) as a regulator of Drosophila Csk (dCsk) activity. dASPP is the homolog of the mammalian ASPP proteins, which are known to bind to and stimulate the proapoptotic function of p53. We show that dASPP is a positive regulator of dCsk. First, dASPP loss-of-function strongly enhances the specific phenotypes of dCsk mutants in wing epithelial cells. Second, dASPP interacts physically with dCsk to potentiate the inhibitory phosphorylation of Drosophila Src (dSrc). Our results suggest a role for dASPP in maintaining epithelial integrity through dCsk regulation.  相似文献   

2.
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.  相似文献   

3.
The C-terminal Src kinase (Csk) family of protein tyrosine kinases contains two members: Csk and Csk homologous kinase (Chk). Both phosphorylate and inactivate Src family kinases. Recent reports suggest that the Src homology (SH) 2 domains of Csk and Chk may bind to different phosphoproteins, which provides a basis for different cellular functions for Csk and Chk. To verify and characterize such a functional divergence, we compared the binding properties of the Csk, Chk, and Src SH2 domains and investigated the structural basis for the functional divergence. First, the study demonstrated striking functional differences between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Second, structural analysis and mutagenic studies revealed that the functional differences among the three SH2 domains were largely controlled by one residue, Glu127 in Csk, Ile167 in Chk, and Lys200 in Src. Mutating these residues in the Csk or Chk SH2 domain to the Src counterpart resulted in dramatic gain of function similar to Src SH2 domain, whereas mutating Lys200 in Src SH2 domain to Glu (the Csk counterpart) resulted in loss of Src SH2 function. Third, a single point mutation of E127K rendered Csk responsive to activation by a Src SH2 domain ligand. Finally, the optimal phosphopeptide sequence for the Chk SH2 domain was determined. These results provide a compelling explanation for the functional differences between two homologous protein tyrosine kinases and reveal a new structure-function relationship for the SH2 domains.  相似文献   

4.
The carboxyl-terminal Src kinase (Csk) is an indispensable negative regulator for the Src family tyrosine kinases (SFKs) that play pivotal roles in various cell signalings. To understand the molecular basis of the Csk-mediated regulation of SFKs, we elucidated the crystal structure of full-length Csk. The Csk crystal consists of six molecules classified as active or inactive states according to the coordinations of catalytic residues. Csk assembles the SH2 and SH3 domains differently from inactive SFKs, and their binding pockets are oriented outward enabling the intermolecular interaction. In active molecules, the SH2-kinase and SH2-SH3 linkers are tightly stuck to the N-lobe of the kinase domain to stabilize the active conformation, and there is a direct linkage between the SH2 and the kinase domains. In inactive molecules, the SH2 domains are rotated destroying the linkage to the kinase domain. Cross-correlation matrices for the active molecules reveal that the SH2 domain and the N-lobe of the kinase domain move as a unit. These observations suggest that Csk can be regulated through coupling of the SH2 and kinase domains and that Csk provides a novel built-in activation mechanism for cytoplasmic tyrosine kinases.  相似文献   

5.
6.
Src kinase regulation by phosphorylation and dephosphorylation   总被引:10,自引:0,他引:10  
Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.  相似文献   

7.
In vitro studies have demonstrated the involvement of Src kinases in several aspects of cell scattering, including cell dissociation and motility. We have therefore sought to explore their functions in the context of the whole organism. Loss-of-function microinjection studies indicate that the ubiquitous Src, Fyn, and Yes tyrosine kinases are specifically implicated in Xenopus gastrulation movements. Injection of mRNAs coding for dominant negative forms of the ubiquitous members of the Src family, namely Fyn, Src, and Yes, perturbs gastrulation movements, resulting in the inability to close the blastopore. Injection of mRNA coding for Csk, a natural inhibitor of Src kinase activity, produces the same phenotypic alterations. The ubiquitous Src kinases have redundant functions in gastrulation movements since overexpression of one member of the family can compensate for the inhibition of another. Interfering mutants of the Src family also inhibit activin-induced morphogenetic movements of animal cap explants isolated from injected embryos. In contrast, these mutants do not interfere with mesoderm induction, as inferred from the presence of mesoderm derivatives and from the expression of early mesodermal markers in injected embryos. In addition, Src kinase activity measured by an in vitro kinase assay is elevated in gastrulating embryos and in FGF- and activin-treated animal caps, confirming the implication of Src enzymatic activity during gastrulation. Altogether, our results demonstrate that Src kinases are essential components of the machinery that drives gastrulation movements independent of mesoderm induction and suggest that Src activity is primarily implicated in cellular movements that take place during the process of cell intercalation.  相似文献   

8.
All members of the Src family of nonreceptor protein tyrosine kinases are phosphorylated and subsequently down-regulated by the C-terminal Src kinase, Csk. Although the recognition of Src protein substrates is essential for a diverse set of signaling events linked to cellular growth and differentiation, the factors controlling this critical protein-protein interaction are not well known. To understand how Csk recognizes Src, the chemical/physical events that modulate apparent substrate affinity and turnover were investigated. Src is phosphorylated in a biphasic manner in rapid quench flow experiments, suggesting that the phosphoryl transfer step is fast and highly favorable and does not limit overall turnover. As opposed to other kinase-substrate pairs, turnover is not limited by the physical release of ADP based on stopped-flow fluorescence and catalytic trapping experiments, suggesting that other steps control net phosphorylation. The K(d) for Src is considerably larger than the K(m) based on single turnover kinetic and equilibrium sedimentation experiments. Taken together, the data are consistent with a mechanism whereby Csk achieves a low K(m) for the substrate Src, not by stabilizing protein-protein interactions but rather by facilitating a fast phosphoryl transfer step. In this manner, the phosphoryl transfer step functions as a chemical clamp facilitating substrate recognition.  相似文献   

9.
10.
Protein-tyrosine kinase C-terminal Src kinase (Csk) was originally purified as a kinase for phosphorylating Src and other Src family kinases. The phosphorylation of a C-terminal tyrosine residue of Src family kinases suppresses their kinase activity. Therefore, most physiological studies regarding Csk function have been focused on Csk as a negative regulator of Src family tyrosine kinases and as a potential tumor suppressor. Paradoxically, the protein levels of Csk were elevated in some human carcinomas. In this report, we show that eukaryotic elongation factor 2 (eEF2) is a new protein substrate of Csk and could locate in the nucleus. We demonstrate that Csk-mediated phosphorylation of eEF2 has no effect on its cytoplasmic function in regulating protein translation. However, phosphorylation of eEF2 enhances its proteolytic cleavage and the nuclear translocation of the cleaved eEF2 through a SUMOylation-regulated process. Furthermore, we show that cleaved fragments of eEF2 can induce nuclear morphological changes and aneuploidy similar to those in cancer cells, suggesting that there is an additional mechanism for Csk in tumorigenesis through regulation of eEF2 subcellular localization.  相似文献   

11.
Csk (carboxyl-terminal Src kinase) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases (SFKs) to inhibit their activities. Recently, we identified a transmembrane protein, Cbp (Csk-binding protein), that, when phosphorylated, can recruit Csk to the membrane where the SFKs are located. The Cbp-mediated relocation of Csk to the membrane may play a role in turning off the signaling events initiated by SFKs. To further characterize the Csk-Cbp interaction, we have generated a reconstituted system using soluble, highly purified proteins. Csk and phosphorylated Cbp were co-purified as a large protein complex consisting of at least four Csk.Cbp units. The addition of the phosphorylated, but not nonphosphorylated, Cbp to an in vitro assay stimulated Csk activity toward Src. Csk was also activated by a phosphopeptide containing the tyrosine in Cbp that binds to Csk (Tyr-314). Kinetic analysis revealed that Cbp or the phosphopeptide induced up to a 6-fold reduction in the K(m) for Src, indicating that the Csk.Cbp complex has a greater affinity for Src than free Csk. These findings suggest that Cbp is involved in the regulation of SFKs not only by relocating Csk to the membrane but also by directly activating Csk.  相似文献   

12.
The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.  相似文献   

13.
Shaffer J  Sun G  Adams JA 《Biochemistry》2001,40(37):11149-11155
The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosphorylation of a substrate peptide, generated from a random library, occurs in two kinetic phases: a rapid, exponential "burst" phase followed by a slow, linear phase. The amplitude of the burst phase increases as a function of enzyme concentration, indicating that the biphasic kinetics are not the result of product inhibition. Analysis of the burst rate as a function of substrate concentration indicates that the phosphoryl transfer step is fast (k3 > or = 140 s(-1) and highly favorable (k3/k-3 > or = 6). The apparent dissociation rate constant for ADP (0.6 s(-1), measured using stopped-flow kinetic methods and a fluorescent trapping agent, mant-ATP, is close to kcat. Since the substrate dissociation constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma-phosphate of ATP to the substrate and rapidly releases the phosphoproduct. Overall rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event in the catalytic cycle is coupled with slow conformational changes.  相似文献   

14.
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.  相似文献   

15.
Regulation of Btk by Src family tyrosine kinases.   总被引:5,自引:1,他引:4       下载免费PDF全文
Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed.  相似文献   

16.
Csk phosphorylates Src family tyrosine kinases and down-regulates their activities in vitro and in vivo. To gain insight into the integrin-mediated cellular functions of this negative regulator of the Src family, we examined integrin-mediated signals in Csk-deficient fibroblasts (Csk(-) cells) and their stable transfectants expressing re-introduced Csk (Csk(-)/Csk cells). Integrin-mediated activation of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase in Csk(-)/Csk cells upon adhesion to fibronectin or laminin-10/11 was down-regulated, whereas Akt activation increased. Interestingly, the suppression of ERK-MAP kinase activation in Csk(-)/Csk cells was restored by overexpression of a dominant-negative Akt. In agreement with these results, Csk(-)/Csk cells were more resistant to apoptosis induced by serum depletion, but were less proliferative, compared with Csk(-) cells. These results, taken together, demonstrate that Csk is an important regulator of integrin-mediated signaling and cellular behavior.  相似文献   

17.
Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.  相似文献   

18.
Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.  相似文献   

19.
Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.  相似文献   

20.
CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.  相似文献   

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