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Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure.

ABSTRACT: 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.

SUBMITTER: Joukov V

PROVIDER: S-EPMC1218276 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Project description: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.

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Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure.

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