title: Design and characterization of an improved protein tyrosine phosphatase substrate-trapping mutant.
abstract: Although members of the protein tyrosine phosphatase (PTPase) family share a common mechanism of action (hydrolysis of phosphotyrosine), the cellular processes in which they are involved can be both highly specialized and fundamentally important. Identification of cellular PTPase substrates will help elucidate the biological functions of individual PTPases. Two types of substrate-trapping mutants are 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 with these two mutants, which are usually relatively abundant cellular proteins. Based on mechanistic considerations, we seek to create novel PTPase mutants with improved substrate-trapping properties. Kinetic and 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 possesses 6- and 28-fold higher substrate-binding affinity than those of D181A and C215S, respectively. In vivo substrate-trapping experiments indicate that D181A/Q262A exhibits much higher affinity than both D181A and 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 newly developed and improved substrate-trapping mutant can serve as a powerful affinity reagent to isolate and purify both high- and low-abundant protein substrates. Given that both Asp181 and Gln262 are invariant among the PTPase family, it is predicted that this improved substrate-trapping mutant would be applicable to all members of PTPases for substrate identification.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Laiping
authors.initials: L
authors.lastname: Xie
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: Zhong-Yin
authors.initials: ZY
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017027:Protein Tyrosine Phosphatases