Role of IRS and PHIP on insulin-induced tyrosine phosphorylation and distribution of IRS proteins

To analyze the functional differences of the insulin receptor substrate ( IRS) family, the N- terminal fragments containing the pleckstrin homology ( PH) domains and the phosphotyrosine- binding ( PTB) domains of IRS ( IRS- N) proteins, as well as intact IRS molecules, were expressed in Cos- 1 cells, and insulin- induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS- N fragments non- selectively inhibited insulin- induced tyrosine phosphorylation of IRS- 1, IRS- 2 and IRS- 3, among which IRS3- N was most effective. The mutations of IRS- 1 disrupting all the phosphoinositide- binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS- 1, which was further inhibited by coexpression of all the IRS- N proteins examined. In contrast, the N- terminal PH domain- interacting region ( PHIP- N) of PH- interacting protein ( PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS- N proteins, but not PHIP- N, suppressed targeting of IRS- 1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity- disrupting mutations of IRS- 1 significantly impaired but did not completely abrogate the insulin- induced translocation of IRS- 1 to the plasma membrane, which was further suppressed by IRS1- N overexpression. These findings suggest that both insulin- induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.