The Second-sphere Residue T263 Is Important For The Function And Activity Of PTP1B

BackgroundThe level of tyrosine phosphorylation in normal cells is fine-tuned by the cooperation of PTPs and protein tyrosine kinases (PTKs). The protein tyrosine phosphatases (PTPs) are important regulator in signal transduction pathways and cell signaling. All classical PTPs share common catalytic mechanisms, and the important catalytic residues in the first sphere of their active sites have been well characterized over the past two decades. However, little attention has been paid to the second-sphere residues that are potentially important in defining the intrinsic activity and substrate specificity of PTPs.ObjectivesTo demonstrate the important roles of the second sphere residue Thr263 of the PTP active site in catalytic mechanism and function of PTP.Materials and MethodsTo test this hypothesis and reveal the underlying molecular mechanism, we performed a detailed study of the wild-type and mutant forms of PTP IB (in which position 263 is substituted) using a combination of cellular, kinetic and crystallographic approaches. The point mutations of PTP1B were generated using pT7-7(PTP1B) or pcDNA4-PTP1B as the template. After HepG2 cells transfected with pcDNA4 or various PTP1B constructs, we tested the negative regulation role of PTP1B and Thr263 mutants in insulin signaling. The wild-type PTP1B and its mutants were expressed in E. coli BL21 and purified by CM-Sephadex C-50 and chromatography. The phosphatase activities of PTP1B and its mutants were determined using pNPP, phosphopeptide as substrates. And a detailed mechanistic study utilizing Br(?)nsted analysis and pH dependence followed the crystallographic studies and structural comparison.ResultsThe T263A, T263S and T263N mutants were functionally impaired mutants and the effects in down-regulating IR phosphorylation were significantly decreased comparing with wild type PTP1B (PTP1B-WT). All tested T263 mutants exhibited lower activity toward both pNPP and phosphopeptide substrates. Thr263 affects the specificity of PTP1B toward certain substrates, such as the IR-phosphopeptide, by directly interacting with residues surrounding the central phosphotyrosine residue. By examining the leaving group dependence and pH profile we can infer that T263 mutants affect both the chemical reaction and the substrate-binding steps and impair PTP1B catalysis by perturbing the function of the general acid. The crystal structures of the T263 mutants of PTP1B suggested that the T263:F182 hydrophobic interaction may be a determinant for phosphatase activity of PTP1B.Conclusions1. The second-sphere residue T263 is important in PTP1B-regulated insulin signaling. Thr263 mutants decreased the effect of PTP1B in down-regulating IR phosphorylation.2. Catalysis in T263-PTP1B mutants is significantly impaired in vitro.3. Thr263 mutants of PTP1B affects both the chemical reaction and the substrate binding steps.4. Thr263 mutants impaired PTP1B catalysis by perturbing the function of the general acid.5. The crystal structures of the T263 mutants of PTP1B suggest that PTP1B T263 could be the determinant to the stabilization of WPD-loop which can modulate the movement of WPD-loop.6. T263:F182 hydrophobic interaction may be a determinant for phosphatase activity of the PTP1B.