DEVELOPMENT OF SUBSTRATE-TRAPPING MUTANTS TO IDENTIFY PHYSIOLOGICAL SUBSTRATES OF PROTEIN-TYROSINE PHOSPHATASES

The identification of substrates of protein tyrosine phosphatases (PTP s) is an essential step toward a complete understanding of the physiol ogical function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues, From a m utagenesis study of these invariant residues that was guided by our kn owledge of the crystal structure of PTP1B, we have discovered a mutati on of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a ''substrate trap.'' Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumul ation of phosphotyrosine primarily on the epidermal growth factor (EGF ) receptor as well as on proteins of 120, 80, and 70 kDa. The associat ion between the D181A mutant of PTP1B and these substrates was suffici ently stable to allow isolation of the complex by immunoprecipitation, As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, f or the EGF receptor, the interaction absolutely requires receptor auto phosphorylation. Furthermore, from immunofluorescence studies, the D18 1A mutant of PTP1B appeared to retain the endogenous EGF receptor in a n intracellular complex, These results suggest that the EGF receptor i s a bona fide substrate for PTP1B in vivo and that one important funct ion of PTP1B is to prevent the inappropriate, ligand-independent, acti vation of newly synthesized EGF receptor in the endoplasmic reticulum, This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phos phatases and the low molecular weight PTPs, Therefore we anticipate th at this method may be widely applicable to facilitate the identificati on of substrates of other members of this enzyme family.