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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
09:40

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Published on: September 28, 2018

Identification of the major SHPTP2-binding protein that is tyrosine-phosphorylated in response to insulin

K Yamauchi1, V Ribon, A R Saltiel

  • 1Department of Physiology and Biophysics, University of Iowa, Iowa City 52242, USA.

The Journal of Biological Chemistry
|July 28, 1995
PubMed

Insights

Insulin signaling involves a tyrosine-phosphorylated 115-kDa protein (pp115) binding to SHPTP2 phosphatase. This interaction is enhanced when SHPTP2 is catalytically inactive, suggesting pp115 is a key substrate.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Signaling

Background:

  • SHPTP2 (Src homology 2 domain-containing protein-tyrosine phosphatase 2) is a key regulator in cellular signaling pathways.
  • Insulin receptor signaling is crucial for glucose homeostasis and metabolic regulation.
  • Tyrosine phosphorylation plays a critical role in mediating signal transduction.

Purpose of the Study:

  • To identify and characterize proteins that associate with SHPTP2 in response to insulin stimulation.
  • To investigate the role of SHPTP2 in insulin signaling and identify its potential substrates.
  • To determine if pp115 is a direct substrate of SHPTP2.

Main Methods:

  • Immunoprecipitation of SHPTP2 from insulin-stimulated cells (3T3L1 adipocytes and CHO cells expressing human insulin receptor).
  • Coimmunoprecipitation to identify associated proteins, followed by SDS-PAGE and Western blotting.
  • Expression of wild-type and catalytically inactive Myc-tagged SHPTP2 mutants to assess functional interactions.

Main Results:

  • Insulin stimulation led to the coimmunoprecipitation of a tyrosine-phosphorylated 115-kDa protein (pp115) with SHPTP2.
  • Platelet-derived growth factor stimulation did not significantly increase pp115 coimmunoprecipitation with SHPTP2, indicating specificity.
  • Association of SHPTP2 with insulin receptor substrate-1 was minimal (<2%).
  • Expression of a catalytically inactive SHPTP2 mutant significantly enhanced pp115 coimmunoprecipitation compared to wild-type SHPTP2.

Conclusions:

  • The insulin-stimulated tyrosine-phosphorylated 115-kDa protein (pp115) is the predominant in vivo SHPTP2-binding protein.
  • pp115 is likely a physiological substrate for the SHPTP2 protein-tyrosine phosphatase.
  • These findings elucidate a novel interaction in insulin signaling, highlighting pp115 as a potential target for SHPTP2 activity.

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