title: Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.
abstract: Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in 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 domains 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 autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
authors.forename: M
authors.initials: M
authors.lastname: Frankel
authors.forename: A J
authors.initials: AJ
authors.lastname: Ablooglu
authors.forename: J W
authors.initials: JW
authors.lastname: Leone
authors.forename: E
authors.initials: E
authors.lastname: Rusinova
authors.forename: J B
authors.initials: JB
authors.lastname: Ross
authors.forename: R L
authors.initials: RL
authors.lastname: Heinrikson
authors.forename: R A
authors.initials: RA
authors.lastname: Kohanski
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013050:Spectrometry, Fluorescence
chemical_list: D016895:Culture Media, Serum-Free; D007328:Insulin; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin