Interaction of the retinal insulin receptor beta-subunit with the p85 subunit of phosphoinositide 3-kinase.
Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable 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 multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.
Rajala
Raju V S
RV
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. raju-rajala@ouhsc.edu
McClellan
Mark E
ME
Chan
Michael D
MD
Tsiokas
Leonidas
L
Anderson
Robert E
RE
D041721:3T3-L1 Cells; D000595:Amino Acid Sequence; D000818:Animals; D007328:Insulin / Q000737:chemistry / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009924:Organ Culture Techniques; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000201:enzymology* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin; D014443:Tyrosine; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C526298:Irs2 protein, mouse; C526299:Irs2 protein, rat; C494920:Akt1 protein, rat