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The COOH-terminal tyrosine phosphorylation sites on IRS-1 bind SHP-2 and negatively regulate insulin signaling
1Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02215, USA. myersmg@joslab.harvard.edu
Abstract:
Activation of tyrosine kinases by numerous growth factor and cytokine receptors leads to tyrosine phosphorylation of the insulin receptor substrate (IRS)-proteins. Tyrosine-phosphorylated motifs on the IRS proteins bind to the SH2 domains in proteins that mediate downstream signals, including phosphatidylinositol 3'-kinase, GRB-2, and SHP-2. We investigated the function of the two SHP-2 binding COOH-terminal tyrosines of IRS-1 by replacing them with phenylalanine (IRS-1(FCT)). IRS-1(FCT) failed to bind SHP-2 or mediate its tyrosine phosphorylation during insulin stimulation. Although several reports suggest a critical role for SHP-2 in insulin stimulated mitogen-activated protein kinase activation and cell proliferation, IRS-1(FCT) mediated these effects normally in 32D cells. Indeed, IRS-1(FCT) exhibited increased tyrosine phosphorylation, phosphatidylinositol 3'-kinase binding and activation of protein synthesis in response to insulin. These results suggest that SHP-2 attentuates the phosphorylation and downstream signal transmission of IRS-1 and that the interaction of IRS-1 and SHP-2 is an important regulatory event which attenuates insulin metabolic responses.
Insights
The SHP-2 protein normally dampens insulin receptor substrate-1 (IRS-1) signaling. Disrupting the IRS-1 and SHP-2 interaction enhances insulin-stimulated protein synthesis and metabolic responses.
Area of Science:
- Molecular biology
- Cell signaling
- Biochemistry
Background:
- Insulin receptor substrate (IRS) proteins are key mediators of insulin signaling.
- Tyrosine phosphorylation of IRS proteins by activated tyrosine kinases initiates downstream signaling cascades.
- SH2 domain-containing proteins, such as SHP-2, bind to phosphorylated IRS proteins to transmit signals.
Purpose of the Study:
- To investigate the role of SHP-2 binding to IRS-1 in insulin signaling.
- To determine the functional consequence of disrupting the interaction between IRS-1 and SHP-2.
Main Methods:
- Site-directed mutagenesis was used to replace the SHP-2 binding tyrosines on IRS-1 with phenylalanine, creating IRS-1(FCT) mutant.
- IRS-1(FCT) was analyzed for its ability to bind SHP-2 and mediate tyrosine phosphorylation.
- Downstream signaling events, including mitogen-activated protein kinase activation, cell proliferation, and protein synthesis, were assessed in response to insulin stimulation.
Main Results:
- The IRS-1(FCT) mutant failed to bind SHP-2 and did not mediate SHP-2 tyrosine phosphorylation upon insulin stimulation.
- Despite reports suggesting SHP-2's role in insulin-stimulated mitogen-activated protein kinase activation and cell proliferation, IRS-1(FCT) supported these processes normally.
- IRS-1(FCT) demonstrated enhanced tyrosine phosphorylation, increased phosphatidylinositol 3'-kinase binding, and augmented protein synthesis activation in response to insulin.
Conclusions:
- SHP-2 acts as a negative regulator, attenuating IRS-1 phosphorylation and downstream signal transmission.
- The interaction between IRS-1 and SHP-2 is a critical regulatory mechanism that dampens insulin's metabolic responses.
- Disrupting the IRS-1/SHP-2 interaction enhances insulin signaling, suggesting a potential target for modulating metabolic outcomes.