Mutation of Thr466 in SHP2 abolishes its phosphatase activity, but provides a new substrate-trapping mutant.
Most classical phosphotyrosyl phosphatases (PTPs), including the Src homology phosphotyrosyl phosphatase 2 (SHP2) possess a Thr or a Ser residue immediately C-terminal to the invariant Arg in the active site consensus motif (H/V-C-X5-R-S/T), also known as the "signature motif". SHP2 has a Thr (Thr466) at this position, but its importance in catalysis has not been investigated. By employing site-directed mutagenesis, phosphatase assays and substrate-trapping studies, we demonstrate that Thr466 is critical for the catalytic activity of SHP2. Its mutation to Ala abolishes phosphatase activity, but provides a new substrate-trapping mutant. We further show that the nucleophilic Cys459 is not involved in substrate trapping by Thr466Ala-SHP2 (T/A-SHP2). Mutation of Thr466 does not cause significant structural changes in the active site as revealed by the trapping of the epidermal growth factor receptor (EGFR), the physiological substrate of SHP2, and by orthovanadate competition experiments. Based on these results and previous other works, we propose that the role of Thr466 in the catalytic process of SHP2 could be stabilizing the sulfhydryl group of Cys459 in its reduced state, a state that enables nucleophilic attack on the phosphate moiety of the substrate. The T/A-SHP2 harbors a single mutation and specifically interacts with the EGFR. Since the nucleophilic Cys459 and the proton donor Asp425 are intact in the T/A-SAHP2, it offers an excellent starting material for solving the structure of SHP2 in complex with its physiological substrate.
Merritt
Rebecca
R
Department of Biochemistry and Molecular Pharmacology and The Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506-9142, USA.
Hayman
Michael J
MJ
Agazie
Yehenew M
YM
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D003545:Cysteine / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000235:genetics*; D041681:NIH 3T3 Cells; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013912:Threonine / Q000235:genetics* / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D047908:Intracellular Signaling Peptides and Proteins; D013912:Threonine; D014638:Vanadates; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; D003545:Cysteine; D000409:Alanine