Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine
Autor: | Michael D. W. Griffin, Thomas E. Smithgall, Bruno Catimel, Ya Chee Lim, Hong-Jian Zhu, Anderly C. Chueh, Mohd Ishtiaq Anasir, Nadia L.Y. Ng, Mai Tran, Daisy Sio Seng Lio, Gahana Advani, Ching-Seng Ang, Heather Verkade, Benjamin E. Turk, Heung-Chin Cheng |
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Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
animal structures Proto-Oncogene Proteins pp60(c-src) lcsh:Medicine Src-family protein kinases Biology SH2 domain Biochemistry 03 medical and health sciences Protein Domains Cell Line Tumor Csk Humans Phosphorylation lcsh:QH573-671 Tyrosine Molecular Biology Binding Sites C-terminal Src kinase Tyrosine-protein kinase CSK lcsh:Cytology Kinase Research lcsh:R HEK 293 cells Tumor suppressor Cell Biology Catalysis colon cancer Cell biology HEK293 Cells 030104 developmental biology Protein kinase domain Mutation Chk Protein Processing Post-Translational Protein Binding Proto-oncogene tyrosine-protein kinase Src |
Zdroj: | Cell Communication and Signaling, Vol 15, Iss 1, Pp 1-22 (2017) Cell Communication and Signaling : CCS |
ISSN: | 1478-811X |
Popis: | 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. Electronic supplementary material The online version of this article (doi:10.1186/s12964-017-0186-x) contains supplementary material, which is available to authorized users. |
Databáze: | OpenAIRE |
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