A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop.
We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.
Noelle
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Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse Cologne, Germany.
Tennagels
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N
Klein
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HW
D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010446:Peptide Fragments; D010710:Phosphates; D010748:Phosphopeptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014443:Tyrosine; D012694:Serine; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; C081827:insulin receptor serine kinase