Phosphorylation of the rodent negative acute-phase protein alpha 1-inhibitor-III by the insulin receptor tyrosine kinase.
alpha 1-Inhibitor-III (alpha 1I3), a broad range proteinase inhibitor, member of the alpha-macroglobulin family, is abundant in normal rat plasma. Insulin-dependent tyrosine phosphorylation of a monomeric 195K glycoprotein (pp195) was observed in wheatgerm agglutinin (WGA)-Sepharose-purified insulin receptor preparations from rat liver and muscle. Phosphorylation of pp195 in vitro required a basic poly-amino acid, i.e. poly-L-lysine. We present evidence identifying pp195 as alpha 1I3. In situ perfusion with saline essentially removed pp195 from rat livers. Addition of normal rat plasma to liver homogenates or to WGA eluates restored insulin-stimulated phosphorylation of pp195; plasma from streptozotocin-diabetic rats was much less effective. Liver-derived pp195 copurified with an abundant plasma protein, with the characteristics of alpha 1I3, on size exclusion and ion-exchange chromatography. An approximately 195K protein, comigrating with alpha 1I3, was markedly diminished in plasma from diabetic rats, and alpha 1I3 concentration was decreased by approximately 70% upon immunoblot analysis. Highly purified alpha 1I3 was phosphorylated by muscle- or liver-derived insulin receptors in the presence of 1 microM poly-L-lysine and comigrated with pp195 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 1I3 phosphorylation was half-maximal at approximately 70 nM and was stimulated by insulin 7-fold. Hindlimb perfusion removed more than 90% plasma albumin but only approximately 20% pp195 from muscles. alpha 1I3 messenger RNA was identified in liver but not in muscle. A specific antibody against alpha 1I3 immunoprecipitated phosphorylated pp195 in WGA-purified insulin receptor preparations from nonperfused liver and from saline perfused and nonperfused muscle. alpha 1I3 is bound and internalized by alpha-macroglobulin receptors; whether it is phosphorylated in vivo is unknown. Hepatic alpha 1I3 synthesis may diminish in diabetic rats.
Komori
K
K
Department of Medicine and Biochemistry/Molecular Biology, Medical University of South Carolina, Charleston 29425.
Robinson
K A
KA
Block
N E
NE
Roberts
R C
RC
Buse
M G
MG
D000209:Acute-Phase Proteins; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D008099:Liver / Q000378:metabolism*; D008297:Male; D009132:Muscles / Q000378:metabolism*; D009928:Organ Specificity; D010477:Perfusion; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*
D000209:Acute-Phase Proteins; D010750:Phosphoproteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; C017894:alpha 1-inhibitor 3; D012313:RNA; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin