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Abstract
The roles of the alpha- and beta-isoforms of phosphatidylinositol (PI) 3'-kinase p85 regulatory subunit were studied with isoform-specific antisera in three model systems in which the insulin receptor mediates rapid phosphorylation of insulin receptor substrate-1 (IRS-1). Insulin receptor signaling stimulated the association of IRS-1 with p85 alpha protein, and p85 alpha-associated PI 3-kinase activity in 3T3-L1 adipocytes, and in transfected Chinese hamster ovary cells (CHO-T) and COS-1 cells expressing high levels of human insulin receptors. While not detectable in 3T3-L1 adipocytes, the p85 beta isoform was also found to associate with IRS-1 in response to insulin receptor activation in COS-1 and CHO-T cells. However, selective immunoprecipitation of p85 beta from unstimulated COS-1 or CHO-T cell lysates was accompanied by higher levels of PI 3-kinase activity than that associated with p85 alpha. Remarkably, the large stimulation of PI 3-kinase activity associated with p85 alpha (7.8 +/- 2.0-fold, n = 6) in insulin-treated CHO-T cells was not observed in p85 beta immunoprecipitates (1.8 +/- 0.6-fold, n = 6), and in COS-1 cells p85 beta-associated PI 3-kinase activity was completely insensitive to stimulation by the insulin receptor. These data suggest the novel hypothesis that binding of p85 beta to IRS-1 complexes in COS-1 and CHO-T cells does not mediate marked activation of PI 3-kinase activity as does p85 alpha.
Highlights
The roles of the a- and p-isoforms of phosphatidyli- cated in mediatinga variety of cellular responses such asminositol (PI) 3”kinase pS5 regulatory subunit were stud- togenic signaling (Fantl et al, 1992; Valius and Kazlauskas, ied with isoform-specific antisera in three model sys- 1993), triggering of secretory pathways such as histamsiencretems in which the insulin receptormediatesrapid tion in basophilic cells (Yano et al, 19931, insulin-stimulated phosphorylationof insulin receptor substrate-1 (IRS-1). glucose transport (Okada et al, 1994; Kanai et al, 1993) and Insulin receptor signaling stimulatedthe associationof Xenopus oocyte maturation (Chuanget al., 1993)
Was found to associate with IRS-1 in response to PI 3-kinase is a heterodimeric enzyme consisting of a catainsulin receptor activation in COS-1andCHO-T cells
The interactionof the p85 regulatory subunit withtyrobinding of p85pto IRS-1 complexesin COS-1and CHO-T sine phosphorylated cognate motifs apparently stimulates incells does not mediate markedactivation of PI 3-kinase trinsic PI 3-kinaseactivity of the enzyme
Summary
ZymoGenetics, 4225 Roosevelt Way NE, Seattle, WA 98105. ll Towhom correspondence should be addressed. Regulation of p8a5nad- p85P-associated PI 3-Kinase pocytes express high levels of IRs and the insulin-regulated whole antisera (20 pl) and preimmune sera (20 pl) to the respective glucose transporter isoform GLUT4 A-Sepharose (20 pl)for 2 h and washedonce in PBSbefore adding the lysates.Sepharose-boundimmune complexes were collected by centrifugation (5000 x g,30 s), washed three times in PB1S%, Nonidet P-40, twice in 500 mM LiCVlOO mM Tris-HC1, pH 7.6, once in 10 mM Tris-HC1, pH 7.4, 100 mM NaCI, 1mM EDTA, and once in PI 3-kinase study IR function These cells lack many of the downstream assay buffer (20mM Tris-HC1, pH 7.4, 100 mM NaCl, 10 mM MgCI,, 0.5 biological target systems for insulin action (e.g. GLUT4), but exhibit early events in insulirneceptor signaling suchas IRS-1 phosphorylation and PI 3-kinase activation (Yonezawa et al, 1992; Backer et al, 1992a, Backeret al., 1992b). Insulin receptor and tyrosine phosphorylated proteins were detected with the monoclonal antibodies CT-1 and PY-20 (Zymed), respectively
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