Abstract
The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.
Highlights
Pendent manner, because of the coordinated action of protein kinases and phosphatases
hematopoietic protein-tyrosine phosphatase (HePTP) is a cytosolic enzyme of hematopoietic origin that is most similar to the brain-specific protein-tyrosine phosphatases (PTPs) STEP and PTP-SL (Fig. 1)
Sequence analysis suggests that the catalytic domains of HePTP, STEP, and PTP-SL possess all of the invariant residues characterizing the PTP family
Summary
Expression and Purification of HePTP and Its Mutants—The coding sequence for the full-length HePTP and the NH2-terminal truncated HePTP/⌬31 were produced by PCR using pGEX-3X-HePTP360 as a template [7]. The PCR product was subjected to KpnI/BamHI digestion and ligated with the HePTP DNA fragment mentioned above, yielding an expression vector for NH2-terminal Histagged HePTP-PTP1B chimera HePTP[1–71]-Gly-Thr-PTP1B(1–321). Determination of Kinetic Parameters Using pNPP, Tyr(P)-containing Peptides, and ERK2/pTpY as Substrates—Kinetic parameters for the PTP-catalyzed hydrolysis of pNPP were determined at 30 °C in pH 7 buffer containing 50 mM 3,3-dimethylglutarate, 1 mM EDTA with an ionic strength of 150 mM adjusted by addition of NaCl as described previously [4]. Because the kcat and Km for HePTP/D236A are much lower than those of the wild-type enzyme, the kinetic parameters for the HePTP/ D236A-catalyzed Tyr(P) peptide hydrolysis were determined using an enzyme-coupled continuous spectrophotometric assay [7]. Kinetic parameters for the dephosphorylation of the phosphorylated ERK2 protein were determined using a continuous spectrophotometric assay described previously [4, 29]
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