Autophosphorylation of the two C-terminal tyrosine residues Tyr1316 and Tyr1322 modulates the activity of the insulin receptor kinase in vitro.
Previously, several studies have demonstrated that autophosphorylation of the C-terminal tyrosine residues (Tyr1316 and Tyr1322) affects the signaling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C-terminal phosphorylation in vitro, we have constructed, purified and characterized 45 kDa soluble insulin receptor kinase domains (IRKD), either with (IRKD) or without (IRKD-Y2F) the two C-terminal tyrosine phosphorylation sites, respectively. According to HPLC phosphopeptide mapping, autophosphorylation of the three tyrosines in the activation loop of the IRKD-Y2F kinase (Tyr1146, Tyr1150, and Tyr1151) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the Km values for exogenous substrates. However, the mutation in IRKD-Y2F resulted in a decrease in the maximum velocities of the phosphotransferase reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD-Y2F led to an increase in the apparent Km values for ATP, suggesting a cross-talk of the C-terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, conformational changes of the enzyme following autophosphorylation were abolished by the removal of the two C-terminal tyrosines. These data suggest a regulatory role of the two C-terminal phosphorylation sites in the phosphotransferase activity of the insulin receptor.
Institute of Biochemistry, University of Cologne, Germany. norbert.tennagels@aventis.com
N
N
Tennagels
E
E
Bergschneider
H
H
Al-Hasani
H W
HW
Klein
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011972:Receptor, Insulin