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1
Nagy, Zoltan, Jun Mori, Vanesa-Sindi Ivanova, Alexandra Mazharian, and Yotis A. Senis. "Interplay between the tyrosine kinases Chk and Csk and phosphatase PTPRJ is critical for regulating platelets in mice." Blood 135, no. 18 (April 30, 2020): 1574–87. http://dx.doi.org/10.1182/blood.2019002848.
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Abstract The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.
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Sabe, H., M. Okada, H. Nakagawa, and H. Hanafusa. "Activation of c-Src in cells bearing v-Crk and its suppression by Csk." Molecular and Cellular Biology 12, no. 10 (October 1992): 4706–13. http://dx.doi.org/10.1128/mcb.12.10.4706.
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The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.
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Sabe, H., M. Okada, H. Nakagawa, and H. Hanafusa. "Activation of c-Src in cells bearing v-Crk and its suppression by Csk." Molecular and Cellular Biology 12, no. 10 (October 1992): 4706–13. http://dx.doi.org/10.1128/mcb.12.10.4706-4713.1992.
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The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.
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Li, Leiming, Masaya Okura, and Akira Imamoto. "Focal Adhesions Require Catalytic Activity of Src Family Kinases To Mediate Integrin-Matrix Adhesion." Molecular and Cellular Biology 22, no. 4 (February 15, 2002): 1203–17. http://dx.doi.org/10.1128/mcb.22.4.1203-1217.2002.
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ABSTRACT Members of the Src family of tyrosine kinases function to phosphorylate focal adhesion (FA) proteins. To explore the overlapping functions of Src kinases, we have targeted Csk, a negative regulator of the Src family, to FA structures. Expression of FA-targeted Csk (FA-Csk) effectively reduced the active form (nonphosphorylated at the C-terminal regulatory tyrosine) of Src members in the cell. We found that fibroblasts expressing FA-Csk lost integrin-mediated adhesion. Activated Src (SrcY529F) as well as activation of putative Src signaling mediators (Fak, Cas, Crk/CrkL, C3G, and Rap1) blocked the effect of FA-Csk in a manner dependent on Rap1. SrcY529F also inhibited activated Ras-induced cell detachment but failed to rescue detachment caused by an activated mutant of Raf1 (Raf-BXB) that Rap1 cannot inhibit. Although normal spreading onto fibronectin was restored by the β1 integrin affinity-activating antibody TS2/16 in cells expressing FA-Csk or Raf-BXB, FAs were lost in these cells. On the other hand, Rap1 activation could restore FAs in cells expressing FA-Csk. Activation of the executioner caspase, caspase 3, is essential for many forms of apoptosis. While a caspase 3 inhibitor (Z-DEVD-FMK) inhibited cell detachment triggered by activation of caspase 8, this inhibitor had no effect on cell detachment caused by FA-Csk. Likewise, overexpression of an activated Akt made cells resistant to the effect of caspase 8 activation, but not to the effect of FA-Csk. It is therefore likely that the primary cause of cell rounding and detachment induced by FA-Csk involves dysfunction of FAs rather than caspase-mediated apoptosis that may result from possible loss of survival signals mediated by Src family kinases. We suggest that endogenous Src family kinases are essential for FAs through activation of Rap1 in fibroblasts.
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Yamaguchi, N., Y. Nakayama, T. Urakami, S. Suzuki, T. Nakamura, T. Suda, and N. Oku. "Overexpression of the Csk homologous kinase (Chk tyrosine kinase) induces multinucleation: a possible role for chromosome-associated Chk in chromosome dynamics." Journal of Cell Science 114, no. 9 (May 1, 2001): 1631–41. http://dx.doi.org/10.1242/jcs.114.9.1631.
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The Csk family of non-receptor-type tyrosine kinases consists of Csk and the Csk homologous kinase Chk. Each enzyme suppresses the catalytic activity of Src family kinases by phosphorylating their C-terminal negative regulatory tyrosine residues. Ectopic and transient expression of Chk in COS-1 cells showed nuclear localization of Chk and growth inhibition. To further explore the role of Chk in cell growth, we overexpressed Chk in human immature myeloid KMT-2 cells. Chk overexpression brought about growth retardation and aberrant chromosome movement leading to multinucleation, and these events were accompanied by insufficient formation of mitotic spindles. In vitro kinase assays showed that Chk overexpression suppressed the tyrosine kinase activity of Lyn, a member of the Src family, immunoprecipitated from Triton X-100 lysates. Subcellular fractionation studies revealed that fractions of Chk and Lyn, resistant to Triton X-100 solubilization, are associated with mitotic chromosome scaffolds and spindles. Chk overexpression induced a decrease in autophosphorylation of Lyn and concomitant changes in levels of tyrosine phosphorylation of proteins associated with both fractions. These results indicate that Chk, Lyn and the tyrosine-phosphorylated proteins localize to mitotic chromosomes and spindles, suggesting that Chk-dependent tyrosine phosphorylation, presumably through Lyn, may be involved in chromosome dynamics.
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Howell, B. W., and J. A. Cooper. "Csk suppression of Src involves movement of Csk to sites of Src activity." Molecular and Cellular Biology 14, no. 8 (August 1994): 5402–11. http://dx.doi.org/10.1128/mcb.14.8.5402.
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Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.
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Howell, B. W., and J. A. Cooper. "Csk suppression of Src involves movement of Csk to sites of Src activity." Molecular and Cellular Biology 14, no. 8 (August 1994): 5402–11. http://dx.doi.org/10.1128/mcb.14.8.5402-5411.1994.
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Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.
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Lee, Byeong-Chel, Shalom Avraham, Akira Imamoto, and Hava Karsenty Avraham. "Identification of the nonreceptor tyrosine kinase MATK/CHK as an essential regulator of immune cells using Matk/CHK-deficient mice." Blood 108, no. 3 (August 1, 2006): 904–7. http://dx.doi.org/10.1182/blood-2005-12-4885.
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Abstract Matk/CHK knockout mice were reported to show no apparent phenotypic abnormalities. This was thought to be due to the homologous kinase Csk that compensates for Matk/CHK. Here, we present the first evidence that the nonreceptor tyrosine kinase, Matk/CHK, is an important modulator of immune cell signaling. We found that the frequency of primitive hematopoietic cells, the side population c-kit+ Lin– Sca-1+ (SPKLS) cells, in Matk/CHK–/– mice was increased 2.2-fold compared with the control mice. Moreover, Matk/CHK deficiency led to significantly higher pre–B cell colony formation following IL-7 stimulation. Interestingly, when mice received the in vivo antigen challenge of TNP-ovalbumin followed by restimulation, the Matk/CHK–/– lymph node and spleen cells produced significantly lower IFN-γ levels compared with the respective wild-type cells. Our study indicates that Matk/CHK is not functionally redundant with Csk, and that this tyrosine kinase plays an important role as a regulator of immunologic responses.
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Chong, Yuh-Ping, Terrence D. Mulhern, and Heung-Chin Cheng. "C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK)—endogenous negative regulators of Src-family protein kinases." Growth Factors 23, no. 3 (January 2005): 233–44. http://dx.doi.org/10.1080/08977190500178877.
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Zagozdzon, Radoslaw, Rafal Kaminski, Yigong Fu, Wei Fu, Cecile Bougeret, and Hava Karsenty Avraham. "Csk homologous kinase (CHK), unlike Csk, enhances MAPK activation via Ras-mediated signaling in a Src-independent manner." Cellular Signalling 18, no. 6 (June 2006): 871–81. http://dx.doi.org/10.1016/j.cellsig.2005.07.016.
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Puthiyaveetil, Sujith, Iskander M. Ibrahim, and John F. Allen. "Evolutionary rewiring: a modified prokaryotic gene-regulatory pathway in chloroplasts." Philosophical Transactions of the Royal Society B: Biological Sciences 368, no. 1622 (July 19, 2013): 20120260. http://dx.doi.org/10.1098/rstb.2012.0260.
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Photosynthetic electron transport regulates chloroplast gene transcription through the action of a bacterial-type sensor kinase known as chloroplast sensor kinase (CSK). CSK represses photosystem I (PS I) gene transcription in PS I light and thus initiates photosystem stoichiometry adjustment. In cyanobacteria and in non-green algae, CSK homologues co-exist with their response regulator partners in canonical bacterial two-component systems. In green algae and plants, however, no response regulator partner of CSK is found. Yeast two-hybrid analysis has revealed interaction of CSK with sigma factor 1 (SIG1) of chloroplast RNA polymerase. Here we present further evidence for the interaction between CSK and SIG1. We also show that CSK interacts with quinone. Arabidopsis SIG1 becomes phosphorylated in PS I light, which then specifically represses transcription of PS I genes. In view of the identical signalling properties of CSK and SIG1 and of their interactions, we suggest that CSK is a SIG1 kinase. We propose that the selective repression of PS I genes arises from the operation of a gene-regulatory phosphoswitch in SIG1. The CSK-SIG1 system represents a novel, rewired chloroplast-signalling pathway created by evolutionary tinkering. This regulatory system supports a proposal for the selection pressure behind the evolutionary stasis of chloroplast genes.
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Neet, K., and T. Hunter. "The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src." Molecular and Cellular Biology 15, no. 9 (September 1995): 4908–20. http://dx.doi.org/10.1128/mcb.15.9.4908.
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CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.
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I. Hussein, Raghad, Zahir M. Hussain, and Salah A. Albermany. "Performance of Differential CSK under Color Noise: A Comparison with CSK." Journal of Engineering and Applied Sciences 15, no. 1 (October 25, 2019): 48–59. http://dx.doi.org/10.36478/jeasci.2020.48.59.
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Wang, Bing, Serge Lemay, Schickwann Tsai, and André Veillette. "SH2 Domain-Mediated Interaction of Inhibitory Protein Tyrosine Kinase Csk with Protein Tyrosine Phosphatase-HSCF." Molecular and Cellular Biology 21, no. 4 (February 15, 2001): 1077–88. http://dx.doi.org/10.1128/mcb.21.4.1077-1088.2001.
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ABSTRACT The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs. This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions. We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation. Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain. Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs. This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells. Surprisingly, the Csk–PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF. Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association. Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain. Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs. Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.
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Drahovska, Z., I. Balicki, A. Trbolova, M. Mihalova, and M. Holickova. "A retrospective study of the occurrence of Chronic Superficial Keratitis in 308 German Shepherd dogs: 1999-2010." Polish Journal of Veterinary Sciences 17, no. 3 (September 1, 2014): 543–46. http://dx.doi.org/10.2478/pjvs-2014-0082.
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Abstract Chronic Superficial Keratitis (CSK) is an autoimmune mediated inflammation of the cornea, that is usually bilateral but often with nonsymmetrical manifestation. The aim of this study was to determine the occurrence and appearance of clinical symptoms of CSK in German Shepherd dogs in Poland and Slovakia. CSK was diagnosed in 308 German Shepherds for a period of 11 years (from 1999 to 2010). The highest incidence of the CSK (p < 0.001) in Slovakia and in Poland was in dogs between the ages of 5-8 years. This study found similarity in gender ratio of affected patients with CSK in two neighboring countries. This disease occurs most often in males, with almost identical frequency in both countries, Slovakia 65.63% and Poland 61.32%, respectively. The incidence of depigmentation and thickening of the external surface of the third eyelid together with CSK was observed in 69.19% of dogs in Poland and in 63.15% of dogs in Slovakia. CSK is often recognized in advanced stage-affected areas with inflammatory process by CSK, obtained frequently from 2 to 3 quadrants.
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Berman-Booty, Lisa D., Rukiye Eraslan, Umesh Hanumegowda, Glenn H. Cantor, Denise I. Bounous, Evan B. Janovitz, Beverly K. Jones, Olesia Buiakova, Michael Hayward, and Susan Wee. "Systemic Loss of C-terminal Src Kinase Expression Elicits Spontaneous Suppurative Inflammation in Conditional Knockout Mice." Veterinary Pathology 55, no. 2 (January 16, 2018): 331–40. http://dx.doi.org/10.1177/0300985817747330.
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C-terminal Src kinase (Csk) is one of the critical negative regulators of the Src family of kinases. The Src family of kinases are nonreceptor tyrosine kinases that regulate inflammation, cell proliferation, motility, and adhesion. To investigate potential histologic lesions associated with systemic loss of Csk gene activity in adult mice, conditional Csk-knockout mice were examined. Cre-mediated systemic excision of Csk induced by tamoxifen treatment resulted in multiorgan inflammation. Specifically, induction of Csk gene excision with three days of tamoxifen treatment resulted in greater than 90% gene excision. Strikingly, these mice developed enteritis that ranged from minimal and suppurative to severe, fibrinonecrosuppurative and hemorrhagic. Other inflammatory lesions included suppurative pneumonia, gastritis, and myocarditis, and increased numbers of inflammatory cells within the hepatic parenchyma. When tamoxifen treatment was reduced from three days to one day in an effort to lower the level of Csk gene excision and limit lesion development, the mice developed severe suppurative to pyogranulomatous pneumonia and minimal to mild suppurative enteritis. Lesions observed secondary to Csk gene excision suggest important roles for Csk in downregulating the proinflammatory activity of the Src family of kinases and limiting neutrophil-mediated inflammation.
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Schmedt, Christian, and Alexander Tarakhovsky. "Autonomous Maturation of α/β T Lineage Cells in the Absence of Cooh-Terminal Src Kinase (Csk)." Journal of Experimental Medicine 193, no. 7 (March 26, 2001): 815–26. http://dx.doi.org/10.1084/jem.193.7.815.
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The deletion of COOH-terminal Src kinase (Csk), a negative regulator of Src family protein tyrosine kinases (PTKs), in immature thymocytes results in the development of α/β T lineage cells in T cell receptor (TCR) β-deficient or recombination activating gene (rag)-1–deficient mice. The function of Csk as a repressor of Lck and Fyn activity suggests activation of these PTKs is solely responsible for the phenotype observed in csk-deficient T lineage cells. We provide genetic evidence for this notion as α/β T cell development is blocked in lck−/−fyn−/− csk-deficient mice. It remains unclear whether activation of Lck and Fyn in the absence of Csk uncouples α/β T cell development entirely from engagement of surface-expressed receptors. We show that in mice expressing the α/β TCR on csk-deficient thymocytes, positive selection is biased towards the CD4 lineage and does not require the presence of major histocompatibility complex (MHC) class I and II. Furthermore, the introduction of an MHC class I–restricted transgenic TCR into a csk-deficient background results in the development of mainly CD4 T cells carrying the transgenic TCR both in selecting and nonselecting MHC background. Thus, TCR–MHC interactions have no impact on positive selection and commitment to the CD4 lineage in the absence of Csk. However, TCR-mediated negative selection of csk-deficient, TCR transgenic cells is normal. These data suggest a differential involvement of the Csk-mediated regulation of Src family PTKs in positive and negative selection of developing thymocytes.
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JOUKOV, Vladimir, Mauno VIHINEN, Satu VAINIKKA, Janusz M. SOWADSKI, Kari ALITALO, and Mathias BERGMAN. "Identification of Csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure." Biochemical Journal 322, no. 3 (March 15, 1997): 927–35. http://dx.doi.org/10.1042/bj3220927.
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The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vivo at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain.
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Radel, C., and V. Rizzo. "Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 2 (February 2005): H936—H945. http://dx.doi.org/10.1152/ajpheart.00519.2004.
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To identify the role of caveolin-1 in integrin mechanotransduction, we exposed bovine aortic endothelial cells to 10 dyn/cm2 of laminar shear stress. Caveolin-1 was acutely and transiently phosphorylated with shear, occurring downstream of β1-integrin activation as the β1-integrin blocking antibody JB1A was inhibitory. In manipulating Src family kinase (SFK) activity with knockdown of Csk or type 1 protein phosphatase (PP1) treatment, we observed coordinate increase and decrease in shear-induced caveolin-1 phosphorylation, respectively. Hence, shear-stimulated caveolin-1 phosphorylation is regulated by SFKs. Shear-induced recruitment and phosphorylation of caveolin-1 occurred at β1-integrin sites in a β1-integrin- and SFK-dependent manner. Csk, described to interact with pY14-caveolin-1 and integrins, bound to an increased pool of phosphorylated caveolin-1 after shear corresponding with elevated Csk at β1-integrin sites. Like caveolin-1, treatment with JB1A and PP1 attenuated shear-induced Csk association with β1-integrins. Csk function was assayed with transfection of a caveolin-1 phosphorylation domain peptide. The peptide attenuated shear-induced association of Csk at β1-integrin sites, as well as colocalization of Csk with paxillin and phosphorylated caveolin-1. Because integrin and Csk activity regulate cytoskeletal reorganization, we evaluated the role of this mechanism in shear-induced myosin light chain (MLC) phosphorylation. Knockdown of Csk expression was sufficient to reduce MLC diphosphorylation due to shear. Disruption of Csk-integrin association by peptide treatment was also inhibitory of the MLC diphosphorylation response. Together these data indicate that integrin activation with shear stress results in SFK-regulated caveolin-1 phosphorylation that, in turn, mediates Csk association at integrin sites, where it plays a role in downstream, shear-stimulated MLC diphosphorylation.
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Duan, Li-Juan, Akira Imamoto, and Guo-Hua Fong. "Dual roles of the C-terminal Src kinase (Csk) during developmental vascularization." Blood 103, no. 4 (February 15, 2004): 1370–72. http://dx.doi.org/10.1182/blood-2003-05-1701.
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Abstract Here we report that C-terminal Src kinase (Csk), a tyrosine kinase that negatively regulates the activity of Src and related kinases, is important for vascular development. In Csk–/– embryos, although vascular tubules were formed and organized into capillary-like networks during the initial genesis of blood vessels, the vessels failed to engage in normal sprout formation. In chimeric embryos containing both wild-type and Csk–/– cells, the presence of wild-type cells enabled Csk–/– endothelial cells to participate in branching morphogenesis. We suggest that wild-type cells may have supplied an angiogenic factor absent in Csk–/– cells. Despite the partial rescue of vascular development in chimeric embryos, the embryos failed to form vitelline vessels and died at E9.5. These results indicate that Csk is required both for angiogenic sprouting and vascular remodeling.
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Honey, Karen. "Csk quenches the fire." Nature Reviews Immunology 4, no. 4 (April 2004): 246. http://dx.doi.org/10.1038/nri1336.
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Dobenecker, Marc-Werner, Christian Schmedt, Masato Okada, and Alexander Tarakhovsky. "The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function." Molecular and Cellular Biology 25, no. 23 (December 1, 2005): 10533–42. http://dx.doi.org/10.1128/mcb.25.23.10533-10542.2005.
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ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.
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Rahmouni, Souad, Torkel Vang, Andres Alonso, Scott Williams, Marianne van Stipdonk, Chiara Soncini, Michel Moutschen, Stephen P. Schoenberger, and Tomas Mustelin. "Removal of C-Terminal Src Kinase from the Immune Synapse by a New Binding Protein." Molecular and Cellular Biology 25, no. 6 (March 15, 2005): 2227–41. http://dx.doi.org/10.1128/mcb.25.6.2227-2241.2005.
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ABSTRACT The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an ∼72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this “parasynaptic” location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.
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Pourati, Jacob, Andrew Maniotis, David Spiegel, Jonathan L. Schaffer, James P. Butler, Jeffrey J. Fredberg, Donald E. Ingber, Dimitrijie Stamenovic, and Ning Wang. "Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?" American Journal of Physiology-Cell Physiology 274, no. 5 (May 1, 1998): C1283—C1289. http://dx.doi.org/10.1152/ajpcell.1998.274.5.c1283.
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We tested the hypothesis that mechanical tension in the cytoskeleton (CSK) is a major determinant of cell deformability. To confirm that tension was present in adherent endothelial cells, we either cut or detached them from their basal surface by a microneedle. After cutting or detachment, the cells rapidly retracted. This retraction was prevented, however, if the CSK actin lattice was disrupted by cytochalasin D (Cyto D). These results confirmed that there was preexisting CSK tension in these cells and that the actin lattice was a primary stress-bearing component of the CSK. Second, to determine the extent to which that preexisting CSK tension could alter cell deformability, we developed a stretchable cell culture membrane system to impose a rapid mechanical distension (and presumably a rapid increase in CSK tension) on adherent endothelial cells. Altered cell deformability was quantitated as the shear stiffness measured by magnetic twisting cytometry. When membrane strain increased 2.5 or 5%, the cell stiffness increased 15 and 30%, respectively. Disruption of actin lattice with Cyto D abolished this stretch-induced increase in stiffness, demonstrating that the increased stiffness depended on the integrity of the actin CSK. Permeabilizing the cells with saponin and washing away ATP and Ca2+ did not inhibit the stretch-induced stiffening of the cell. These results suggest that the stretch-induced stiffening was primarily due to the direct mechanical changes in the forces distending the CSK but not to ATP- or Ca2+-dependent processes. Taken together, these results suggest preexisting CSK tension is a major determinant of cell deformability in adherent endothelial cells.
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YAQUB, Sheraz, Hilde ABRAHAMSEN, Bastian ZIMMERMAN, Natalya KHOLOD, Knut Martin TORGERSEN, Tomas MUSTELIN, Friedrich W. HERBERG, Kjetil TASKÉN, and Torkel VANG. "Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain." Biochemical Journal 372, no. 1 (May 15, 2003): 271–78. http://dx.doi.org/10.1042/bj20030021.
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In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser364 by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.
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Mulyadi, Hanung Agus, Augy Syahailatua, and Zainal Arifin. "THE CO-OPERATIVE STUDY OF KUROSHIO (CSK): IS IT BENEFICIAL FOR INDONESIA?" Marine Research in Indonesia 44, no. 2 (November 7, 2019): 63–71. http://dx.doi.org/10.14203/mri.v44i2.562.
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The Cooperative Study of Kuroshio and its marginal seas (CSK) is one of the international joint research project conducted in the Western Pacific region. Many Asian countries had been involved in this project from 1965 to1979. Data and information from the CSK are enormous and cover wide-ranging aspects of marine science from the Kuroshio and adjacent regions (e.g. physical aspects, biological aspects and biogeochemical aspects). Indonesia had committed to participate actively in several marine research programs in the area linked to the CSK program by conducting marine research in its internal waters. This essay explained the CSK from biological aspects and Indonesia perspective. During the CSK, biological aspects (e.g. primary productivity, zooplankton biomass, and fisheries) were studied intensively. Indonesia conducted research in internal waters (Natuna Sea and the Java Sea) for oceanography monitoring and fish stock assessment. Participation in the CSK program allowed Indonesia to pursue the establishment of the National Center for Ocean Research (NCOR), develop human capacity building, research properties and standardized all techniques and procedures related to oceanography aspects. After the CSK, Indonesia has continued to conduct marine research linked to the previous study. We learn a lot from the past CSK that a key to succeeding in running this program depending on co-operative spirit, enthusiastic in understanding marine science from the region and enhancing human capacity for doing better marine research.
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Deng, Linhong, Nigel J. Fairbank, Ben Fabry, Paul G. Smith, and Geoffrey N. Maksym. "Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells." American Journal of Physiology-Cell Physiology 287, no. 2 (August 2004): C440—C448. http://dx.doi.org/10.1152/ajpcell.00374.2003.
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Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in cells. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20–L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456–L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferrimagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 ± 1.6% of the beads were associated with actin structures compared with only 6.7 ± 2.8% before MS and 38.4 ± 5.5% in time-matched controls ( P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KCl or 10−4 M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma.
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Rathore, Vipul B., Peter J. Newman, and Debra K. Newman. "Paxillin Becomes Tyrosine-Phosphorylated and Binds Csk in a GpIIb-IIIa-Dependent Manner Following GPVI-Induced Activation of Murine Platelets." Blood 104, no. 11 (November 16, 2004): 3889. http://dx.doi.org/10.1182/blood.v104.11.3889.3889.
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Though Src family kinases (SFKs) play a critical role in collagen-induced platelet activation, little is known about how SFK activity is regulated following exposure of platelets to collagen. In resting cells, SFKs are maintained in an inactive conformation, in part, via intramolecular interactions between their SH2 domain and a C-terminal tyrosine residue whose phosphorylation state is controlled by the C-terminal Src kinase, Csk. Access of Csk to SFKs, in turn, is regulated by recruitment of Csk, via its SH2 domain, to one or more tyrosine-phosphorylated Csk-binding proteins, which include Csk binding protein (Cbp/PAG) itself, paxillin, and its closely-related paxillin family member, Hic-5. Recent studies have shown that human platelets possess only two Csk-binding proteins: Cbp/PAG and Hic-5, and that Hic-5 can become tyrosine-phosphorylated when platelets are stimulated with a variety of platelet agonists, including, thrombin, U46619, and collagen. The purpose of the present investigation was to characterize the complement of Csk-binding proteins in murine platelets and to begin to determine their role in regulating collagen-induced platelet activation. Murine platelets, like human platelets, were found to express Hic-5, and in addition contained two other Csk-binding proteins: paxillin and leupaxin. Of these, both paxillin and Hic-5 became tyrosine phosphorylated, and paxillin was shown to be able to recruit Csk in a time-dependent manner following exposure of murine platelets to collagen and the GPVI/FcRg chain collagen receptor-specific agonist, CRP. These data suggest that the function of Hic-5 in human platelets may be performed by both Hic-5 and paxillin in mice. Finally, both paxillin tyrosine phosphorylation and Csk recruitment were blocked by agents that interfered with either platelet granule secretion or with integrin engagement, consistent with the notion that members of the paxillin family function as integrin-dependent negative feedback regulators of platelet adhesion and spreading.
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Hata, A., H. Sabe, T. Kurosaki, M. Takata, and H. Hanafusa. "Functional analysis of Csk in signal transduction through the B-cell antigen receptor." Molecular and Cellular Biology 14, no. 11 (November 1994): 7306–13. http://dx.doi.org/10.1128/mcb.14.11.7306.
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In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.
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Bergman, M., V. Joukov, I. Virtanen, and K. Alitalo. "Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading." Molecular and Cellular Biology 15, no. 2 (February 1995): 711–22. http://dx.doi.org/10.1128/mcb.15.2.711.
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The C-terminal Src kinase p50csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not significantly suppressed, while the amount of p50csk and Csk activity increased at least 10-fold during 3 days of induction. Immunofluorescence for the induced p50csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the alpha v beta 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These phenotypic changes occurred in several clones analyzed and were totally reversible when Csk was switched off, but they did not occur in cells overexpressing the catalytically inactive Csk R-222 mutant or luciferase. Our results thus show that a fraction of cellular Csk is targeted to focal adhesions via its SH2 and SH3 domains, probably interacting with tyrosyl-phosphorylated focal adhesion proteins. They also suggest that Csk is involved in the regulation of integrins controlling cell attachment and shape.
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Rathore, Vipul B., Masato Okada, Peter J. Newman, and Debra K. Newman. "Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets." Biochemical Journal 403, no. 2 (March 26, 2007): 275–81. http://dx.doi.org/10.1042/bj20061618.
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SFKs (Src family kinases) contribute importantly to platelet function in haemostasis. SFK activity is controlled by Csk (C-terminal Src kinase), which phosphorylates a C-terminal tyrosine residue on SFKs, resulting in inhibition of SFK activity. Csk is recruited to sites of SFK activity by tyrosine-phosphorylated Csk-binding proteins. Paxillin, a multidomain adaptor protein, has been shown to act as a Csk-binding protein and to inhibit Src activity during growth factor signalling. Human platelets express Hic-5, a member of the paxillin family; however, its ability to act as a Csk-binding protein has not been characterized. We sought to identify and characterize the ability of paxillin family members to act as Csk-binding proteins during platelet activation. We found that murine and human platelets differ in the complement of paxillin family members expressed. Human platelets express Hic-5, whereas murine platelets express paxillin and leupaxin in addition to Hic-5. In aggregating human platelets, Hic-5 was tyrosine phosphorylated and recruited Csk via its SH2 domains. In aggregating murine platelets, however, Csk bound preferentially to paxillin, even though both paxillin and Hic-5 were abundantly present and became tyrosine phosphorylated. The SFK Lyn, but not Src or Fyn, was associated with paxillin family members in resting and aggregated human and murine platelets. Lyn, however, was phosphorylated on its C-terminal inhibitory tyrosine residue only following platelet aggregation, which was coincident with recruitment of Csk to paxillin and/or Hic-5 in a manner dependent on prior αIIbβ3 engagement. These observations support the notion that Hic-5 and paxillin function as negative feedback regulators of SFKs in aggregated platelets and that, when both are present, paxillin is preferentially used.
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Hata, A., H. Sabe, T. Kurosaki, M. Takata, and H. Hanafusa. "Functional analysis of Csk in signal transduction through the B-cell antigen receptor." Molecular and Cellular Biology 14, no. 11 (November 1994): 7306–13. http://dx.doi.org/10.1128/mcb.14.11.7306-7313.1994.
Full textAbstract:
In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.
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Vang, Torkel, Knut Martin Torgersen, Vibeke Sundvold, Manju Saxena, Finn Olav Levy, Bjørn S. Skålhegg, Vidar Hansson, Tomas Mustelin, and Kjetil Taskén. "Activation of the Cooh-Terminal Src Kinase (Csk) by Camp-Dependent Protein Kinase Inhibits Signaling through the T Cell Receptor." Journal of Experimental Medicine 193, no. 4 (February 19, 2001): 497–508. http://dx.doi.org/10.1084/jem.193.4.497.
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In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor–CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 ζ chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on ζ chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.
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Bougeret, Cécile, Shuxian Jiang, Iafa Keydar, and Hava Avraham. "Functional Analysis of Csk and CHK Kinases in Breast Cancer Cells." Journal of Biological Chemistry 276, no. 36 (July 9, 2001): 33711–20. http://dx.doi.org/10.1074/jbc.m104209200.
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Martínez-Ciro, Roger Alexander, Francisco Eugenio López-Giraldo, Andrés Felipe Betancur-Perez, and Jose Martín Luna-Rivera. "Design and Implementation of a Multi-Colour Visible Light Communication System Based on a Light-to-Frequency Receiver." Photonics 6, no. 2 (April 11, 2019): 42. http://dx.doi.org/10.3390/photonics6020042.
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Colour-shift keying (CSK) is a visible light communication (VLC) modulation scheme used in the existing IEEE 802.15.7 standard. In CSK, information is transmitted by changing the light intensities of the RGB LEDs. In this work, a low-complexity VLC system is proposed using CSK modulation and a novel receiver based on a light-to-frequency (LTF) converter. At the receiver, CSK symbols are interpreted and decoded in terms of frequencies, which are processed by a counter module of a generic microcontroller, thus avoiding the use of analog-to-digital converters (ADCs), which results in a low-cost VLC system. The main contributions of this work are summarized in the following key points: (1) A low-complexity receiver for CSK modulation is introduced; (2) A particle swarm optimization (PSO) algorithm for CSK constellation design is suggested considering the restrictions of the LTF based receiver; (3) Experimental and theoretical validation is perfomed for the proposed multi-colour VLC system. The results show that this system can provide a transmission speed of 100 kbps using a 4-CSK-LTF constellation for a symbol error rate (SER) of 10 − 4 and a signal to noise ratio (SNR) around 35 dB. These results suggest that the analysed system could find applications on those scenarios where low transmission speeds and ease of deployment are the goals.
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Davidson, Dominique, Lionel M. L. Chow, and André Veillette. "Chk, a Csk Family Tyrosine Protein Kinase, Exhibits Csk-like Activity in Fibroblasts, but Not in an Antigen-specific T-cell Line." Journal of Biological Chemistry 272, no. 2 (January 10, 1997): 1355–62. http://dx.doi.org/10.1074/jbc.272.2.1355.
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Vuica, Milena, Stephen Desiderio, and Jonathan P. Schneck. "Differential Effects of B Cell Receptor and B Cell Receptor–FcγRIIB1 Engagement on Docking of Csk to GTPase-activating Protein (GAP)-associated p62." Journal of Experimental Medicine 186, no. 2 (July 21, 1997): 259–67. http://dx.doi.org/10.1084/jem.186.2.259.
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The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcγ receptors of the IIB1 type (FcγRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcγRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein–associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab′)2 resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcγRIIB1 engagement on this association was abolished by blockade of FcγRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcγRIIB1-deficient cell line IIA1.6 and recovered when FcγRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcγRIIB1–mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor–mediated signals in B cells.
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Xu, Shengli, Jianxin Huo, Joy En-Lin Tan, and Kong-Peng Lam. "Cbp Deficiency Alters Csk Localization in Lipid Rafts but Does Not Affect T-Cell Development." Molecular and Cellular Biology 25, no. 19 (October 1, 2005): 8486–95. http://dx.doi.org/10.1128/mcb.25.19.8486-8495.2005.
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ABSTRACT The ubiquitously expressed transmembrane adaptor Csk-binding protein (Cbp) recruits Csk to lipid rafts, where the latter exerts its negative regulatory effect on the Src family of protein tyrosine kinases. We have inactivated Cbp in the mouse germ line. In contrast to Csk gene inactivation, which leads to embryonic lethality and impaired T-cell development, Cbp-deficient mice were viable and exhibited normal T-cell development but with an increased thymocyte population. In the absence of Cbp, the amount of Csk that localizes to the lipid rafts was greatly reduced. Interestingly, this altered lipid raft localization of Csk did not lead to any detectable biochemical or functional defect in T cells. The T-cell receptor-induced intracellular calcium flux, cell proliferation, and cytokine secretion were not affected by the absence of Cbp. Peripheral T-cell tolerance to superantigen SEB was also largely intact in Cbp-deficient mice. Thus, Cbp is dispensable for T-cell development and activation.
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Murphy, S. M., M. Bergman, and D. O. Morgan. "Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 9 (September 1993): 5290–300. http://dx.doi.org/10.1128/mcb.13.9.5290.
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The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.
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Musso, T., L. Varesio, X. Zhang, T. K. Rowe, P. Ferrara, J. R. Ortaldo, J. J. O'Shea, and D. W. McVicar. "IL-4 and IL-13 induce Lsk, a Csk-like tyrosine kinase, in human monocytes." Journal of Experimental Medicine 180, no. 6 (December 1, 1994): 2383–88. http://dx.doi.org/10.1084/jem.180.6.2383.
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Lsk is a protein tyrosine kinase with homology to the COOH-terminal Src kinase (Csk). Unlike Csk that is ubiquitously expressed, Lsk has limited tissue distribution. Here we have examined the expression and regulation of Lsk and Csk in peripheral human monocytes. We have found that Lsk mRNA and protein were not expressed in resting monocytes but were induced by treatment with interleukin 4 (IL-4) or IL-13 but not by interferon gamma (IFN-gamma) or IL-2. In fact, IFN-gamma, but not IL-2, efficiently blocked Lsk induction by IL-4 or IL-13. In contrast, Csk was constitutively present in human monocytes and was upregulated by IFN-gamma but not by IL-4 or IL-13. These results suggest that despite their structural similarities, Lsk and Csk may play distinct regulatory roles in monocyte functions elicited by cytokines, with Lsk functioning specifically within the context of a Th2-type immune response.
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Murphy, S. M., M. Bergman, and D. O. Morgan. "Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 9 (September 1993): 5290–300. http://dx.doi.org/10.1128/mcb.13.9.5290-5300.1993.
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The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.
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Chan, Khai-Chew, Daisy Sio-Seng Lio, Renwick C. J. Dobson, Boonyarin Jarasrassamee, Mohammed I. Hossain, Aainaa K. Roslee, Kim K. Ia, Matthew A. Perugini, and Heung-Chin Cheng. "Development of the procedures for high-yield expression and rapid purification of active recombinant Csk-homologous kinase (CHK): Comparison of the catalytic activities of CHK and CSK." Protein Expression and Purification 74, no. 2 (December 2010): 139–47. http://dx.doi.org/10.1016/j.pep.2010.07.007.
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Kunte, Dhananjay P., Ramesh K. Wali, Jennifer L. Koetsier, Anna Y. Kilimnik, and Hemant K. Roy. "C-TERMINAL Src KINASE (CSK)." American Journal of Gastroenterology 99 (October 2004): S330. http://dx.doi.org/10.14309/00000434-200410001-00998.
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Stamenović, Dimitrije, and Ning Wang. "Invited Review: Engineering approaches to cytoskeletal mechanics." Journal of Applied Physiology 89, no. 5 (November 1, 2000): 2085–90. http://dx.doi.org/10.1152/jappl.2000.89.5.2085.
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An outstanding problem in cell biology is how cells sense mechanical forces and how those forces affect cellular functions. Various biophysical and biochemical mechanisms have been invoked to answer this question. A growing body of evidence indicates that the deformable cytoskeleton (CSK), an intracellular network of interconnected filamentous biopolymers, provides a physical basis for transducing mechanical signals into biochemical signals. Therefore, to understand how mechanical forces regulate cellular functions, it is important to know how cells respond to changes in the CSK force balance and to identify the underlying mechanisms that control transmission of mechanical forces throughout the CSK and bring it to equilibrium. Recent developments of new experimental techniques for measuring cell mechanical properties and novel theoretical models of cellular mechanics make it now possible to identify and quantitate the contributions of various CSK structures to the overall balance of mechanical forces in the cell. This review focuses on engineering approaches that have been used in the past two decades in studies of the mechanics of the CSK.
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Tobe, K., H. Sabe, T. Yamamoto, T. Yamauchi, S. Asai, Y. Kaburagi, H. Tamemoto, et al. "Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins." Molecular and Cellular Biology 16, no. 9 (September 1996): 4765–72. http://dx.doi.org/10.1128/mcb.16.9.4765.
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Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.
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Davidson, Dominique, Burkhart Schraven, and André Veillette. "PAG-Associated FynT Regulates Calcium Signaling and Promotes Anergy in T Lymphocytes." Molecular and Cellular Biology 27, no. 5 (January 1, 2007): 1960–73. http://dx.doi.org/10.1128/mcb.01983-06.
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ABSTRACT Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.
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Vielreicher, Martin, Gregory Harms, Elke Butt, Ulrich Walter, and Achim Obergfell. "Dynamic Interaction between Src and C-terminal Src Kinase in Integrin αIIbβ3-mediated Signaling to the Cytoskeleton." Journal of Biological Chemistry 282, no. 46 (September 12, 2007): 33623–31. http://dx.doi.org/10.1074/jbc.m704107200.
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Integrin-bound Src tyrosine kinase mediates αIIbβ3 out-side-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during αIIbβ3-mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2–3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Csk-mediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.
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Dey, Nandini, Pradip K. De, Mu Wang, Hongying Zhang, Erika A. Dobrota, Kent A. Robertson, and Donald L. Durden. "CSK Controls Retinoic Acid Receptor (RAR) Signaling: a RAR-c-SRC Signaling Axis Is Required for Neuritogenic Differentiation." Molecular and Cellular Biology 27, no. 11 (February 26, 2007): 4179–97. http://dx.doi.org/10.1128/mcb.01352-06.
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ABSTRACT Herein, we report the first evidence that c-SRC is required for retinoic acid (RA) receptor (RAR) signaling, an observation that suggests a new paradigm for this family of nuclear hormone receptors. We observed that CSK negatively regulates RAR functions required for neuritogenic differentiation. CSK overexpression inhibited RA-mediated neurite outgrowth, a result which correlated with the inhibition of the SFK c-SRC. Consistent with an extranuclear effect of CSK on RAR signaling and neurite outgrowth, CSK overexpression blocked the downstream activation of RAC1. The conversion of GDP-RAC1 to GTP-RAC1 parallels the activation of c-SRC as early as 15 min following all-trans-retinoic acid treatment in LA-N-5 cells. The cytoplasmic colocalization of c-SRC and RARγ was confirmed by immunofluorescence staining and confocal microscopy. A direct and ligand-dependent binding of RAR with SRC was observed by surface plasmon resonance, and coimmunoprecipitation studies confirmed the in vivo binding of RARγ to c-SRC. Deletion of a proline-rich domain within RARγ abrogated this interaction in vivo. CSK blocked the RAR-RA-dependent activation of SRC and neurite outgrowth in LA-N-5 cells. The results suggest that transcriptional signaling events mediated by RA-RAR are necessary but not sufficient to mediate complex differentiation in neuronal cells. We have elucidated a nongenomic extranuclear signal mediated by the RAR-SRC interaction that is negatively regulated by CSK and is required for RA-induced neuronal differentiation.
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Sefercik, U. G., N. Yastikli, and C. Atalay. "URBAN MODELLING PERFORMANCE OF NEXT GENERATION SAR MISSIONS." ISPRS - International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences XLII-2/W7 (September 13, 2017): 641–46. http://dx.doi.org/10.5194/isprs-archives-xlii-2-w7-641-2017.
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In synthetic aperture radar (SAR) technology, urban mapping and modelling have become possible with revolutionary missions TerraSAR-X (TSX) and Cosmo-SkyMed (CSK) since 2007. These satellites offer 1m spatial resolution in high-resolution spotlight imaging mode and capable for high quality digital surface model (DSM) acquisition for urban areas utilizing interferometric SAR (InSAR) technology. With the advantage of independent generation from seasonal weather conditions, TSX and CSK DSMs are much in demand by scientific users. The performance of SAR DSMs is influenced by the distortions such as layover, foreshortening, shadow and double-bounce depend up on imaging geometry. In this study, the potential of DSMs derived from convenient 1m high-resolution spotlight (HS) InSAR pairs of CSK and TSX is validated by model-to-model absolute and relative accuracy estimations in an urban area. For the verification, an airborne laser scanning (ALS) DSM of the study area was used as the reference model. Results demonstrated that TSX and CSK urban DSMs are compatible in open, built-up and forest land forms with the absolute accuracy of 8&ndash;10&thinsp;m. The relative accuracies based on the coherence of neighbouring pixels are superior to absolute accuracies both for CSK and TSX.
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Smida, Michal, Anita Posevitz-Fejfar, Vaclav Horejsi, Burkhart Schraven, and Jonathan A. Lindquist. "A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation." Blood 110, no. 2 (July 15, 2007): 596–625. http://dx.doi.org/10.1182/blood-2006-07-038752.
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Abstract In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.