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Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.

Abstract：

PURPOSE:To identify the tyrosine-phosphorylated protein(s) in bovine rod outer segments (ROS) that are associated with phosphatidylinositol 3-kinase (PI3K). METHODS:Glutathione-S-transferase (GST) fusion proteins containing two SH2 domains of the p85 regulatory subunit of PI3K-GST-p85 (N-SH2), GST-p85 (C-SH2), and respective SH2 mutants (N-SH2, R358A, and C-SH2, R649A)-were prepared and used to pull down tyrosine-phosphorylated proteins in bovine ROS. Protein identity was established by Western blot analysis. PI3K activity was determined in the pull-down mixtures and in immunoprecipitates by incubation with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) and [gamma(32)P]adenosine triphosphate (ATP). RESULTS:The GST pull-down assays indicated the binding of a 97-kDa protein by GST-p85 (N-SH2) in tyrosine-phosphorylated (PY)-ROS that was not present in nonphosphorylated (N)-ROS. Binding was completely abolished when the Arg 358 in the N-SH2 domain was mutated to Ala. Increased binding of the p110alpha catalytic subunit to GST-p85 (N-SH2) fusion protein was also observed in the presence of the 97-kDa phosphorylated protein. Biochemical evidence indicated that the 97-kDa protein was the beta-subunit of the insulin receptor beta-subunit (IRbeta). Immunoprecipitates of PY-ROS and N-ROS with anti-PY antibodies, probed with anti-IRbeta, indicated the presence of IRbeta only in PY-ROS. Immunoprecipitates of PY-ROS and N-ROS with anti-IRbeta antibodies, probed with anti-p85 and anti-p110alpha antibodies, indicated increased amounts of both p85 and p110alpha in PY-ROS compared to N-ROS. Treatment of ROS with insulin, followed by immunoprecipitation with either anti-IRbeta or anti-PY, resulted in increased PI3K activity. Expression and phosphorylation of the cytoplasmic tail of retina insulin receptor showed direct involvement with the p85 subunit of PI3K in vitro. CONCLUSIONS:Tyrosine phosphorylation of the beta-subunit of the insulin receptor is involved in the regulation of PI3K activity in ROS.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Rajala RV,Anderson RE

keywords:

subject

Has Abstract

pub_date

2001-12-01 00:00:00

pages

3110-7

issue

eissn

0146-0404

issn

1552-5783

journal_volume

pub_type

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Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.