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Src kinase activity is regulated by the SHP-1 protein-tyrosine phosphatase
A K Somani1, J S Bignon, G B Mills
1Department of Medicine, University of Toronto and the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Abstract:
Activation of the cellular Src tyrosine kinase depends upon dephosphorylation of the carboxyl-terminal inhibitory tyrosine phosphorylation site. Herein we show that Src isolated from human platelets and Jurkat T cells is preferentially dephosphorylated at its inhibitory phosphotyrosine site by the SHP-1 tyrosine phosphatase. The data also revealed association of Src with SHP-1 in both platelets and lymphocytes and the capacity of Src to phosphorylate SHP-1 and interact with the SHP-1 NH2-terminal SH2 domain in vitro. Analysis of Src activity in thymocytes from SHP-1-deficient motheaten and viable motheaten mice revealed this kinase activity to be substantially lower than that detected in wild-type thymocytes, but to be enhanced by in vitro exposure to SHP-1. Similarly, immunoblotting analysis of thymocyte Src expression before and after selective depletion of active Src protein indicated that the proportion of active relative to inactive Src protein is markedly reduced in motheaten compared with wild-type cells. These observations, together with the finding of reduced Src activity in HEY cells expressing a dominant negative form of SHP-1, provide compelling evidence that SHP-1 functions include the positive regulation of Src activation.
Insights
The tyrosine phosphatase SHP-1 dephosphorylates and activates the cellular Src tyrosine kinase. SHP-1 deficiency in mice leads to reduced Src kinase activity, highlighting SHP-1
Area of Science:
- Molecular Biology
- Cellular Signaling
- Immunology
Background:
- Cellular Src (c-Src) tyrosine kinase activation requires dephosphorylation of its inhibitory tyrosine site.
- The role of specific tyrosine phosphatases in c-Src regulation is not fully elucidated.
Purpose of the Study:
- To investigate the role of SHP-1 tyrosine phosphatase in the activation of cellular Src tyrosine kinase.
- To determine the interaction between SHP-1 and c-Src in human platelets and lymphocytes.
Main Methods:
- Isolation and analysis of Src from human platelets and Jurkat T cells.
- In vitro studies of Src-SHP-1 interaction and phosphorylation.
- Analysis of Src activity in thymocytes from SHP-1-deficient (motheaten) and wild-type mice.
- Immunoblotting to assess Src protein activation status.
- Studies in HEY cells expressing dominant-negative SHP-1.
Main Results:
- SHP-1 preferentially dephosphorylates the inhibitory site of Src in human platelets and T cells.
- Src and SHP-1 associate in platelets and lymphocytes; Src can phosphorylate SHP-1.
- SHP-1-deficient thymocytes exhibit significantly lower Src activity compared to wild-type.
- Src activity is restored in vitro by SHP-1 exposure in deficient cells.
- Reduced proportion of active Src in motheaten thymocytes compared to wild-type.
- Dominant-negative SHP-1 expression leads to reduced Src activity.
Conclusions:
- SHP-1 tyrosine phosphatase positively regulates the activation of cellular Src tyrosine kinase.
- SHP-1 plays a critical role in controlling Src kinase activity in lymphocytes and platelets.