Tyrosine phosphorylation of tub and its association with Src homology 2 domain-containing proteins implicate tub in intracellular signaling by insulin

A mutation in the tub gene leads to maturity-onset obesity, insulin resista nce, and progressive retinal and cochlear degeneration in mice. tub is a me mber of a growing family of genes that encode proteins of unknown function that are remarkably conserved across species. The absence of obvious transm embrane domain(s) or signal sequence peptide motif(s) suggests that Tub is an intracellular protein. Additional sequence analysis revealed the presenc e of putative tyrosine phosphorylation motifs and Src homology 2 (SH2)bindi ng sites. Here we demonstrate that in CHO-IR cells, transfected Tub is phos phorylated on tyrosine in response to insulin and insulin-like growth facto r-1 and that in PC12 cells, insulin but not EGF induced tyrosine phosphoryl ation of endogenous Tub. In vitro, Tub is phosphorylated by purified insuli n receptor kinase as well as by Abl and JAK 2 but not by epidermal growth f actor receptor and Src kinases, Furthermore, upon tyrosine phosphorylation, Tub associated selectively with the SH2 domains of Abl, Lck, and the C-ter minal SH2 domain of phospholipase Cy and insulin enhanced the association o f Tub with endogenous phospholipase Cy in CHO-IR cells. These data suggest that Tub may function as an adaptor protein linking the insulin receptor, a nd possibly other protein-tyrosine kinases, to SH2-containing proteins.