Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor

Receptor tyrosine kinases may use intrasteric inhibition to suppress autoph osphorylation prior to growth factor stimulation. To test this hypothesis w e made an Asp1161Ala mutant in the activation loop that relieved intrasteri c inhibition of the unphosphorylated insulin receptor (IR) and its recombin ant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated confor mational changes and greater catalytic efficiency from a 10-fold increase i n k(cat) and a 15-fold-lower K-m (ATP) although K-m peptide was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase do mains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autoph osphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was n ot constitutively autophosphorylated. However, it displayed a 2.5-fold-lowe r 50% effective concentration for insulin stimulation of autophosphorylatio n and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal sta te, these results demonstrate that neither intrasteric inhibition against A TP binding nor suppression of kinase activity is required to prevent premat ure autophosphorylation of the IR. Finally, the lower rate of dephosphoryla tion suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.