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Activation of Src family kinase activity by the G protein-coupled thrombin receptor in growth-responsive fibroblasts
Y H Chen1, J Pouysségur, S A Courtneidge
1Centre de Biochimie, CNRs UMR134, Nice, France.
Abstract:
Thrombin stimulates G protein-coupled signaling pathways in target cells by proteolytic cleavage of its seven transmembrane domain receptor. Protein tyrosine phosphorylation is also stimulated by the protease via poorly defined mechanisms. In human platelets, thrombin has been shown to activate the nonreceptor tyrosine kinase Src. To elucidate the signal transduction pathways involved in transmission of thrombin's cellular effects, we have examined the ability of thrombin to activate Src family tyrosine kinases in a growth-responsive line of lung fibroblasts (CCL39 cells). We report here that thrombin induces a rapid (< or = 30 s) and transient increase in the kinase activity of Src and Fyn as determined by autophosphorylation in immune complex kinase assays. Activation is mediated by the G protein-coupled thrombin receptor since a synthetic peptide agonist of the receptor mimics thrombin action. The involvement of one or more G proteins in this response was confirmed by the observation that thrombin's effect is partially sensitive to pertussis toxin. Furthermore, both alpha 2-adrenergic and muscarinic m1 receptors are able to increase Src kinase activity via pertussis toxin-sensitive and -insensitive G proteins, respectively. These findings suggest that nonreceptor tyrosine kinases of the Src family may represent a novel effector system linking G protein-coupled receptors to downstream activation of Ras and the mitogen-activated protein kinase cascade.
Insights
Thrombin rapidly activates Src and Fyn tyrosine kinases in lung fibroblasts via the G protein-coupled receptor pathway. This suggests a novel link between G protein-coupled receptors and downstream signaling cascades like Ras/MAPK.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- Thrombin activates G protein-coupled receptors (GPCRs) and protein tyrosine phosphorylation through poorly understood mechanisms.
- In platelets, thrombin activates the Src nonreceptor tyrosine kinase.
- Understanding thrombin's signaling pathways is crucial for elucidating cellular responses.
Purpose of the Study:
- To investigate thrombin's ability to activate Src family tyrosine kinases in lung fibroblasts (CCL39 cells).
- To elucidate the signal transduction pathways mediating thrombin's cellular effects.
- To determine if GPCRs link to Src family kinases.
Main Methods:
- Immune complex kinase assays to measure Src and Fyn autophosphorylation.
- Utilizing a synthetic peptide agonist for the G protein-coupled thrombin receptor.
- Assessing the effect of pertussis toxin on thrombin-induced kinase activation.
Main Results:
- Thrombin rapidly (<30s) and transiently increased Src and Fyn kinase activity.
- Activation was mediated by the G protein-coupled thrombin receptor.
- The response was partially sensitive to pertussis toxin, indicating involvement of G proteins.
- Other GPCRs (alpha 2-adrenergic, muscarinic m1) also activated Src kinases via pertussis toxin-sensitive and -insensitive G proteins.
Conclusions:
- Src family kinases are rapidly activated by thrombin through its GPCR.
- This activation involves G proteins, some of which are pertussis toxin-sensitive.
- Nonreceptor tyrosine kinases of the Src family may link GPCRs to downstream effectors like Ras and MAPK.
- This identifies a novel signaling pathway connecting GPCRs to mitogenic signaling cascades.