Interaction of the human insulin receptor tyrosine kinase from the baculovirus expression system with protein kinase C in a cell-free system.

J Ahn,D.B Donner,O.M Rosen

doi:10.1016/s0021-9258(18)53213-0

Abstract

The cytoplasmic domain of the human insulin receptor (HIR) from the baculovirus expression system (BIRK) is a soluble, constitutively activated protein-tyrosine kinase. In a cell-free system, BIRK is phosphorylated on serine and threonine residues by protein kinase C (PKC) purified from rat brain. Two-dimensional tryptic phosphopeptide mapping of PKC-phosphorylated BIRK identified one phosphothreonine and three phosphoserine peptides, which were also in tryptic digests of insulin receptors from insulin- or PMA-treated Chinese hamster ovary (CHO) cells transfected with the HIR. After Lys-C proteolysis of PKC-phosphorylated BIRK, radioactive phosphopeptides were purified on a C8 reverse-phase high pressure liquid chromatography column. Amino acid sequence analysis identified a phosphothreonine peptide corresponding to amino acids 1331-1340 of the HIR. This peptide contains only one threonine, amino acid 1336, which is identified as a site for PKC phosphorylation in BIRK. CHO cells transfected with the wild type (CHO/HIR) or a mutant human insulin receptor (CHO/HIRT1336N), in which threonine 1336 was substituted with asparagine, were 32P labeled and then stimulated with insulin or phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide analysis of the HIR revealed that phosphorylation of phosphothreonine peptide T, shown to be in PKC-phosphorylated BIRK, was increased by insulin or PMA. However, the corresponding peptide was not in the mutant receptor. Therefore, the present study directly identifies threonine 1336 in the HIR as a phosphorylation site for insulin and PMA. These data also show that BIRK can be used as a model for the study of the regulation of the insulin receptor kinase.

Highlights

From the Memorial Sloan-Kettering Cancer Center and the Sloan-Kettering Division of the Graduate School of Medical Sciences, Cornell Uniuersity, New York,New York 10021
The requirement sional tryptic phosphopeptide mapping of PKC-phos- of tyrosine kinase activity for insulin action [5, 6] and the phorylated baculovirusinsulin receptorkinase (BIRK) identified one phosphothreonineand observation that uncontrolled tyrosine kinase activnistyome three phosphoserine peptides, whichwere in tryptic digests of insulin receptors from insulin-or PMAtreated Chinese hamster ovary(CHO)cells transfected with the HIR
The processes that regulate the receptor tyrosine kinase are incompletely understood; it has beensuggested that phosphorylations by serine/threoninekinasesattenuate it.s activity [8, 9]

Summary

Purification of the BaculovirusInsulin Receptor Kinase and Protein

30% glycerol a t -70 "C for 1year without significant loss of tyrosine kinase activity toward histone H2b. Phosphorylation of BIRK by Protein Kinase C-BIRK (1.25 pg) was incubated with PKC (4 units)a t 30 "C in 25 mM Tris-HCI The reaction was started by the addition of PKC (20 units) and [y3'P]ATP (2,000 cpm/pmol) and was terminated after 30 min by the addition of 110 pl of ice-cold 100%trichloroacetic acid. Phosphorylated receptors were purified by (lane a1;,437cpm) was comparable to thatin BIRK incubated with PKC (laneb; 2,275 cpm). Phospholipids together with Ca2+ further increased BIRK chromatography on wheat germ agglutinin-agarose and immunopre- phosphorylation by PKC (19,280 cpm, lane f ) 9.5-fold above cipitated with the anti-HIR monoclonal antibody CII-25.3 [22]. The autophosphorylation level (2,036cpm, lane e ) .The phosphorylation of BIRK by PKC was saturable and time- and dose-dependent (data not shown), indicating that BIRK is a substrate for PKC in this reconstituted cell-free system

RESULTS

Tyrosi nRee ceptorInsulin

DISCUSSION

Peptide C with a syntheticpeptidewiththeamino sequence around