Departmental Bulletin Paper Critical role of the length of the first β strand in insulin receptor kinase activity

Bolormaa, Enkhtuvshin  ,  Hiroyuki, Tamemoto  ,  Shuichi, Nagashima  ,  Bayasgalan, Tumenbayar  ,  Kent, Sakai  ,  Jun-ichi, Osuga  ,  Manabu, Takahashi  ,  Shin-ichi, Tominaga  ,  Shun, Ishibashi

BackgroundMutations in the tyrosine kinase domain of the insulin receptor gene cause a monogenic syndrome ofinsulin resistance in humans. The first β strand in this kinase domain is close to the nucleotide bindingloop, and its length is conserved through several species. To test whether the exact length of this region isessential for the kinase activity of the insulin receptor, we constructed a one-amino-acid deletion mutant inthe first β strand of the tyrosine kinase domain.MethodsDeletion of Arg1000 with concomitant substitution of Glu1001 to Gln (R1000E1001→ Q) was generated by sitedirectedmutagenesis. Chinese hamster ovary cells were transfected with mutant or wild-type humaninsulin receptor cDNA, and stable clones with similar binding activity were screened by insulin bindingassay and used for further experiments. Receptor expression, kinase activity and downstream insulinsignaling were examined by western blot analysis.ResultsMature insulin receptor expression was comparable between the wild-type and mutant cells. The mutantinsulin receptor showed markedly defective tyrosine kinase activity. Akt kinase phosphorylation wasseverely reduced, indicating that downstream insulin signaling was also impaired by the mutant receptor.ConclusionThis study suggests that the deletion of one residue in the first β strand results in a distortion of theoptimal positioning of the kinase structure, thereby compromising the kinase activity of the

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