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The C-terminal Src inhibitory kinase (Csk)-mediated tyrosine phosphorylation is a novel molecular mechanism to limit P2X3 receptor function in mouse sensory neurons.

ABSTRACT: On sensory neurons, sensitization of P2X(3) receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X(3) receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X(3) receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X(3) receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X(3) receptor function and outlines a potential new target for trigeminal pain suppression.

SUBMITTER: D'Arco M

PROVIDER: S-EPMC2755864 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The C-terminal Src inhibitory kinase (Csk)-mediated tyrosine phosphorylation is a novel molecular mechanism to limit P2X3 receptor function in mouse sensory neurons.

D'Arco Marianna M Giniatullin Rashid R Leone Vanessa V Carloni Paolo P Birsa Nicol N Nair Asha A Nistri Andrea A Fabbretti Elsa E

The Journal of biological chemistry 20090609 32

On sensory neurons, sensitization of P2X(3) receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X(3) receptor, we investigated how Csk might regulate receptor act ...[more]

PMID: 19509283

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Project description: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.

Dataset Information

The C-terminal Src inhibitory kinase (Csk)-mediated tyrosine phosphorylation is a novel molecular mechanism to limit P2X3 receptor function in mouse sensory neurons.

Publications

The C-terminal Src inhibitory kinase (Csk)-mediated tyrosine phosphorylation is a novel molecular mechanism to limit P2X3 receptor function in mouse sensory neurons.

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