| Abstract
| - Although members of the protein tyrosine phosphatase (PTPase) family share a commonmechanism of action (hydrolysis of phosphotyrosine), the cellular processes in which they are involvedcan be both highly specialized and fundamentally important. Identification of cellular PTPase substrateswill help elucidate the biological functions of individual PTPases. Two types of substrate-trapping mutantsare being used to isolate PTPase substrates. In the first, the active site Cys residue is replaced by a Ser(e.g., PTP1B/C215S) while in the second, the general acid Asp residue is substituted by an Ala (e.g.,PTP1B/D181A). Unfortunately, only a limited number of PTPase substrates have been identified withthese two mutants, which are usually relatively abundant cellular proteins. Based on mechanisticconsiderations, we seek to create novel PTPase mutants with improved substrate-trapping properties. Kineticand thermodynamic characterization of the newly designed PTP1B mutants indicates that PTP1B/D181A/Q262A displays lower catalytic activity than that of D181A. In addition, D181A/Q262A also possesses6- and 28-fold higher substrate-binding affinity than those of D181A and C215S, respectively. In vivosubstrate-trapping experiments indicate that D181A/Q262A exhibits much higher affinity than both D181Aand C215S for a bona fide PTP1B substrate, the epidermal growth factor receptor. Moreover, D181A/Q262A can also identify novel, less abundant substrates, that are missed by D181A. Thus, this newlydeveloped and improved substrate-trapping mutant can serve as a powerful affinity reagent to isolate andpurify both high- and low-abundant protein substrates. Given that both Asp181 and Gln262 are invariantamong the PTPase family, it is predicted that this improved substrate-trapping mutant would be applicableto all members of PTPases for substrate identification.
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