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Abstract
We have shown previously that insulin stimulated the tyrosine phosphorylation of the alpha-type 85-kDa subunit (p85) of phosphatidylinositol (PI) 3-kinase in vitro and in vivo. In the present work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor. [32P]Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis. The tyrosine phosphorylation sites of alpha-type p85 in vivo were assigned to three major phosphopeptides, designated p1, p2, and p3. Highly purified insulin receptor also phosphorylated the purified p85 of PI 3-kinase from the bovine thymus at p1. The purified glutathione S-transferase (GST)-p85 (alpha-type) fusion protein and its truncated proteins from Escherichia coli were also phosphorylated by the purified insulin receptor at p1, p2, and p3 in vitro. Analysis of [32P]phosphopeptide of the truncated GST-p85 (alpha-type) fusion proteins and radiosequence analysis revealed that the p1, p2, and p3 phosphopeptides were phosphorylated at tyrosines 607, 580, and 368, respectively. In addition, phenylalanine substitutions at tyrosine 607 and 580 reduced the p1 and p2 phosphopeptides in vivo, respectively. We conclude that the alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo.
Highlights
Hideki Hayashi$, Yasuhiko NishiokaS,Seika KamoharaS, FumihikKo anaiS, Kazuo IshiiS, Yasuhisa Fukuis, Futoshi Shibasakill, Tadaomi Takenawaq, HirosKhid011,Nobuhiko Katsunumall, and Yousuke Ebina$**
We identified the tyrosine kinase activity is essential for manyif not all of the major tyrosine phosphorylation siteosf the a-type p85 biological effects of insulin.theexact molecular bactfayrnyh-ortaroytmolhsypmielzenyaeiesntdpyops8lughue5lronsiansidpinpnohghrpeyoiecrranpeeyntptvlaidatedcocttratirhesos.leielnn-s[pd3slaaihi2tgfyaPeteesse]serPrteihiolhonoenfisscguptahrhlo-oiotfnppypephpetrhorpefeorotpieasrds8tpmemih5ssao.eniTorncnybhteltveaawitliveineqoderuedidphi3mnah-eskvouciernhlyinaanlsonacetitsaibmouaencsseetnitslvoiiniadttkyehirinnaentprgaiefinridgeetuicdi-lnepap(cth6rtioooe)sarn. pRskehoeifocnitencaynesprtleollh-yusmo,liasnierptedhmihaaiaemtttseiadmdbbyeuotlenlyininorcoofposspriiueantncotehidlpwp(itPthhaaIoyat)sest*-s were assigned to three major phosphopeptides, desig- of cells overexpressing the human insulin receptor, nated p l, p2, and p3
The cDNAsencoding p85 have been its truncated proteins fromEscherichia coli were cloned from mouse, human, and bovine, and it has become phosphorylated by the purified insulin receptor a t p l, apparent of the existence of at least two types of p85, desigp2, and p3in vitro.Analysis of [32P]phosphopeptideof nated a and 8 [17,18,19]
Summary
Human insulin receptorswere purified fromCHO cells overexpressing io Rotention limo (mln) humaninsulinreceptorsusing ananti-(human insulin receptor) FIG.. C, regions of the bovine p85 applied to a C18 reverse-phase HPLC column in 0.1:; trifluoroacetic acid and developed with a 0-60rh linear gradient of acetonitrile for 60 min a t a flow rate of 0.5 ml/min. 0.5 mM isopropyl-1-thio-13-D-galactopyranosidTe.he cells were lysed and the GST-p85 (a-type) (whole and deleted) fusion proteins were purified by using a glutathione-Sepharose 4B column, as described under"Experimental Procedures." To the purifiedfusion proteins (about 3 p g ) we added 1 P M [yR'P]ATP and antireceptor antibodypurified insulin receptors in the presence of 10" M insulin, as described under"Experimental Procedures."After 30 min a t room temperature, the kinase reaction was halted by addition of 2 X SDS sample buffer, analyzed on a 8% SDS-PAGE. Thce llulose plate was electrophoresed in 1%ammonium carbonate, pH 8.9, a t 800 V until the marker dye
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