FIGURE 4.

Activation loop residues contribute to the kinase specificity of Irfin1. A, activation loop sequences of insulin receptor-related (IRR) and IR/IGF-1R are aligned with analogous residues from the 10 most closely related human kinases. Arrowheads and residue numbers indicate the residues that are the subject of the mutagenesis experiments. B, inhibition of IR by Irfin1 in cell lysates. Lysates of CHO-K1 cells expressing wild type IR were pretreated (or not, as indicated) with 10 μm Irfin1 and stimulated with insulin. IR autophosphorylation was monitored by Western blotting for phospho-IR (pIR). C, the experiment in B was repeated for wild type and mutant IR constructs and quantified. Each set of bars was normalized to the vehicle-treated, insulin-stimulated phospho-IR Western blot signal. Error bars represent the S.E. from three replicates.