Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine
Abstract Background C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-08-01
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| Series: | Cell Communication and Signaling |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12964-017-0186-x |
| _version_ | 1818265829195644928 |
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| author | Gahana Advani Ya Chee Lim Bruno Catimel Daisy Sio Seng Lio Nadia L. Y. Ng Anderly C. Chüeh Mai Tran Mohd Ishtiaq Anasir Heather Verkade Hong-Jian Zhu Benjamin E. Turk Thomas E. Smithgall Ching-Seng Ang Michael Griffin Heung-Chin Cheng |
| author_facet | Gahana Advani Ya Chee Lim Bruno Catimel Daisy Sio Seng Lio Nadia L. Y. Ng Anderly C. Chüeh Mai Tran Mohd Ishtiaq Anasir Heather Verkade Hong-Jian Zhu Benjamin E. Turk Thomas E. Smithgall Ching-Seng Ang Michael Griffin Heung-Chin Cheng |
| author_sort | Gahana Advani |
| collection | DOAJ |
| description | Abstract Background C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. Methods We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. Results Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. Conclusions SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells. |
| first_indexed | 2024-12-12T19:57:01Z |
| format | Article |
| id | doaj.art-1eea7f5ad1d14905a145ead4e484aea3 |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-12-12T19:57:01Z |
| publishDate | 2017-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-1eea7f5ad1d14905a145ead4e484aea32022-12-22T00:13:50ZengBMCCell Communication and Signaling1478-811X2017-08-0115112210.1186/s12964-017-0186-xCsk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosineGahana Advani0Ya Chee Lim1Bruno Catimel2Daisy Sio Seng Lio3Nadia L. Y. Ng4Anderly C. Chüeh5Mai Tran6Mohd Ishtiaq Anasir7Heather Verkade8Hong-Jian Zhu9Benjamin E. Turk10Thomas E. Smithgall11Ching-Seng Ang12Michael Griffin13Heung-Chin Cheng14Department of Biochemistry & Molecular Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneWalter and Eliza Hall Institute for Medical Research and Department of Medical Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneWalter and Eliza Hall Institute for Medical Research and Department of Medical Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneDepartment of Surgery, University of Melbourne, Royal Melbourne HospitalDepartment of Pharmacology, Yale University School of MedicineDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of MedicineBio21 Biotechnology and Molecular Science Institute, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneDepartment of Biochemistry & Molecular Biology, University of MelbourneAbstract Background C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. Methods We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. Results Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. Conclusions SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.http://link.springer.com/article/10.1186/s12964-017-0186-xSrc-family protein kinasesTumor suppressorCatalysis, colon cancerC-terminal Src kinaseCskChk |
| spellingShingle | Gahana Advani Ya Chee Lim Bruno Catimel Daisy Sio Seng Lio Nadia L. Y. Ng Anderly C. Chüeh Mai Tran Mohd Ishtiaq Anasir Heather Verkade Hong-Jian Zhu Benjamin E. Turk Thomas E. Smithgall Ching-Seng Ang Michael Griffin Heung-Chin Cheng Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine Cell Communication and Signaling Src-family protein kinases Tumor suppressor Catalysis, colon cancer C-terminal Src kinase Csk Chk |
| title | Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine |
| title_full | Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine |
| title_fullStr | Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine |
| title_full_unstemmed | Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine |
| title_short | Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine |
| title_sort | csk homologous kinase chk is an efficient inhibitor of src family kinases but a poor catalyst of phosphorylation of their c terminal regulatory tyrosine |
| topic | Src-family protein kinases Tumor suppressor Catalysis, colon cancer C-terminal Src kinase Csk Chk |
| url | http://link.springer.com/article/10.1186/s12964-017-0186-x |
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