Interaction of the Retinal Insulin Receptor -Subunit with the P85 Subunit of Phosphoinositide 3-Kinase

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• 作者：Raju V. S. Rajala ; Mark E. McClellan ; Michael D. Chan ; Leonidas Tsiokas ; and Robert E. Anderson
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：May 18, 2004
• 年：2004
• 卷：43
• 期：19
• 页码：5637 - 5650
• 全文大小：373K
• 年卷期：v.43,no.19(May 18, 2004)
• ISSN：1520-4995

文摘

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivothrough light activation of the insulin receptor beta2.gif" BORDER=0 ALIGN="middle">-subunit. In this study, we have cloned the 41 kDacytoplasmic region of the retinal insulin receptor (IRbeta2.gif" BORDER=0 ALIGN="middle">) and used the two-hybrid assay of protein-proteininteraction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunitof PI3K and the cytoplasmic region of IRbeta2.gif" BORDER=0 ALIGN="middle">. Under conditions where IRbeta2.gif" BORDER=0 ALIGN="middle"> autophosphorylates, substitutionof Y1322F and M1325P in IRbeta2.gif" BORDER=0 ALIGN="middle"> resulted in the abolition of p85 binding to the IRbeta2.gif" BORDER=0 ALIGN="middle">, confirming that thep85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta2.gif" BORDER=0 ALIGN="middle"> was also confirmed in the yeastthree-hybrid system. Using the C-terminal region of IRbeta2.gif" BORDER=0 ALIGN="middle"> (amino acids 1293-1343 encompassing theYHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined thatthe IRbeta2.gif" BORDER=0 ALIGN="middle">-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulinactivation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assayswith p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, iscapable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin,IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiplesignaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.