Effects of tyrosine-->phenylalanine mutations on auto- and trans-phosphorylation reactions catalyzed by the insulin receptor beta-subunit cytoplasmic domain.
Activation of the insulin receptor kinase is closely associated with autophosphorylation of several tyrosine residues in the cytoplasmic domain of the receptor's two beta-subunits. To determine the contribution of these tyrosine phosphorylations to autoactivation of the receptor kinase, we have blocked phosphorylation at specific tyrosine by replacing these tyrosine residues, individually and in combination, with phenylalanine in a soluble 45-kD analog of the cytoplasmic insulin receptor kinase domain (CIRK). Kinetic studies of auto- and transphosphorylation with this panel of mutated CIRKs indicate that: (i) None of the tyrosines (953, 960, 1,146, 1,150, 1,151, 1,316, or 1,322) are necessary for catalysis: all single Y-->F mutants retain the ability to autoactivate comparable to the parent CIRK. (ii) Two of the tyrosine autophosphorylation sites, either tyrosine 1,150 or 1,151, contribute most (70-80%) of the autoactivation, because replacement of these two tyrosines by phenylalanine was the minimal change that abolishes autoactivation. (iii) A mutant CIRK having all seven reported tyrosine phosphorylation sites replaced by phenylalanine retained basal kinase activity but was incapable of autoactivation. These findings imply that autoactivation can occur without phosphorylation having occurred at any single site (953, 960, 1,146, 1,150, 1,151, 1,316, or 1,322), and autophosphorylation need not follow an ordered, sequential pathway beginning, for example, at tyrosine 1,146 as proposed for the intact insulin receptor.
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6059.
J E
JE
Smith
Z F
ZF
Sheng
R G
RG
Kallen
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D010649:Phenylalanine; D011972:Receptor, Insulin