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Abstract
The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase.
Highlights
Theregulation of kinaseactivity associated with that this receptor exists ainheterotetrameric disulfide-linked insulin receptor by phosphorylation and dephosphocomplex containing two M,= 125,000 a and two M, = 90,000 rylation has beenexaminedusingpartiallypurified
Subsequent studies demonstrated that insuallisno increased the phosphorylation of the insulin receptor in rat hepatocytes ( 7 ),human placenta (8-101, 3T3-Ll adipocytes achieved by prior incubationof receptor withconcen- ( 8 ),and rat adipocytes (11).In cell-free systems, the interactrations of ATP approaching 1 mM
2, and 3).Phosphotyrosine and phosphoserine residuesincrease in the level of phosphoserine with the simultaneous are present in these three domains while peak2 con- appearance of phosphotyrosine (6), whereas the increase by tains phosphothreonine as well
Summary
10 1984 by The Amencan Society of Biological Chemists, Inc. Tyrosine Phosphorylation of the Insulin Receptor/3 Subunit Activates the Receptor-associated Tyrosine KinaseActivity*. Subsequent studies demonstrated that insuallisno increased the phosphorylation of the insulin receptor in rat hepatocytes ( 7 ) ,human placenta (8-101, 3T3-Ll adipocytes achieved by prior incubationof receptor withconcen- ( 8 ) ,and rat adipocytes (11).In cell-free systems, the interactrations of ATP approaching 1 mM. Petruzzelli et al (8) demonstrated that insulin activated a Incubation of the phosphorylatedinsulinreceptor with alkaline phosphatase a1t 5 “C results in theselective dephosphorylationof the phosphotyrosine residues on the fi subunit of the receptor while thephosphoserine and phosphothreonine contents are not affected. The labeling of the p subunit of the insulin rosine content in peak 2 , while very small decreases receptorwithphotoactive ATP analogues provided further are observedinpeaks 1 and 3, respectively.These support for the notion that the insulinreceptor may possess results demonstrate that the exteonf tphosphorylation intrinsic kinase activity (14-16). These studies stronglysuggest that the insulinreceptor functions as a protein kinase
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