BackgroundC-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.MethodsWe 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.ResultsOur 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.ConclusionsSFKs 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 materialThe online version of this article (doi:10.1186/s12964-017-0186-x) contains supplementary material, which is available to authorized users.

No abstract

No abstract

No abstract

Crk SH3 domain binding guanine nucleotide release protein (C3G), Rap guanine nucleotide exchange factor 1 (RAPGEF1), Guanine nucleotide-releasing factor 2 (GRF2) 620 C3G 622 C3G C3G 623 C Downregulated Fernandez et al. 2008 Lymphoproliferative disorder Mutation, Y572C Parker et al. 2016 Human prostate carcinoma Regulates PLCg1-mediated adhesion Peak et al. 2008 Juvenile neuronal ceroid lipofuscinosis Upregulated Lebrun et al. 2011 624 C3G fate determinant. While its catalytic activity enables activation of small GTPases of Ras family, it also has cellular roles independent of its catalytic activity. The fact that other Rap1 GEFs do not compliment C3G during mammalian embryonic development suggests important noncatalytic functions, or its ability to activate other GTPases. Recent studies have shown that altered C3G levels are associated with human cancers and other disorders, suggesting its role in maintaining homeostasis in adult tissues.