IRS PLECKSTRIN HOMOLOGY DOMAINS BIND TO ACIDIC MOTIFS IN PROTEINS

Using a yeast two-hybrid system, we identified several proteins that i nteract with the PH domains in IRS-1 and IRS-2, including Lon protease , myeloblast protein, and nucleolin, Although the roles of these molec ules in insulin action are not yet known, each protein contained an ac idic motif that interacted with the PH domain of IRS-2. However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH d omain in IRS-1 and IRS-2 are not identical. Moreover, synthetic peptid es based on the acidic motif in Lon protease and myeloblast protein in hibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains . The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in beta ARK, phospho lipase C gamma, or spectrin did not bind nucleolin. In 32D cells, nucl eolin bound to both IRS-1 and IRS-2, and expression of the acidic moti f of nucleolin inhibited insulin-stimulated tyrosine phosphorylation o f IRS-1 and IRS-2, These results suggest that the binding of acidic mo tifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the acti vated insulin receptor. Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic pep tide motifs in membrane proteins or other acidic membrane elements tha t couple IRS proteins to activated membrane receptors.