Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.
A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.
Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
J M
JM
Tavaré
B
B
Clack
L
L
Ellis
D000595:Amino Acid Sequence; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin