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Autophosphorylation of the two C‐terminal tyrosine residues Tyr 1316 and Tyr 1322 modulates the activity of the insulin receptor kinase in vitro
Author(s) -
Tennagels Norbert,
Bergschneider Eva,
Al-Hasani Hadi,
Klein Helmut W
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01879-2
Subject(s) - autophosphorylation , phosphorylation , biochemistry , insulin receptor , tyrosine , phosphopeptide , phosphotransferase , receptor tyrosine kinase , chemistry , sh2 domain , biology , insulin , protein kinase a , insulin resistance , endocrinology
Previously, several studies have demonstrated that autophosphorylation of the C‐terminal tyrosine residues (Tyr 1316 and Tyr 1322 ) 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 (Tyr 1146 , Tyr 1150 , and Tyr 1151 ) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the K m 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 K m 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.