Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

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 Thr 183 and Tyr 185 in the activation loop. Tyr 185 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 k cat /K m of 2.6 ؋ 10 6 M ؊1 s ؊1 . In contrast, the k cat /K m 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 Thr 106 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 (ϳ20-fold increase in k cat /K m ) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (ϳ20-fold increase in k cat /K m ) by organizing the catalytic residues with respect to Tyr(P) 185 for optimal phosphoryl transfer.The mitogen-activated protein (MAP) 1 kinases are central components in cellular signaling (1-3). The extent and duration of MAP kinase activation in response to extracellular stimuli are tightly regulated in a cell type-and stimulus-dependent manner, because of the coordinated action of protein kinases and phosphatases. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the activation loop by specific MAP kinase kinases. Downregulation of MAP kinase activity is carried out by multiple protein phosphatases, including Ser/Thr-specific, Tyr-specific, and dual specificity phosphatases (4). This would inevitably lead to the formation of monophosphorylated MAP kinases. Indeed, recent evidence indicates that both forms of the monophosphorylated extracellular signal-regulated kinase 2 (ERK2), a founding member of the MAP kinase family, exist in living cells, in addition to the bisphosphorylated and unphosphorylated forms (5, 6). Interestingly, the activity of the monophosphorylated ERK2/pY and ERK2/pT is ϳ2 and 3 orders of magnitude higher than that of the unphosphorylated ERK2 and is only 1 and 2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY (7). ...

Structure of the Hematopoietic Tyrosine Phosphatase (HePTP) Catalytic Domain: Structure of a KIM Phosphatase with Phosphate Bound at the Active Site

“…This observation is supported by kinetic studies of HePTP that demonstrated that the catalytic activity of HePTP towards phosphorylated substrates increased by one to two orders of magnitude when Thr106 was mutated to either aspartic acid or asparagine. 31 Because this increase in catalytic efficiency was observed towards only phosphorylated peptides and not towards the biphosphorylated Erk2 protein (Erk2/pTpY), Thr106 has been postulated to function as a negative determinant to restrict HePTP catalytic activity towards HePTP-specific substrates. 31 Second, the PTP1B peptide recognition loop residue Arg47 interacts extensively with both the backbone and side-chains of the bound EGFR peptide residues.…”

MAP kinases and the control of nuclear events

“…For example, hematopoietic protein-tyrosine phosphatase (HePTP), mitogen-activated protein kinase phosphatase-3 (MKP3), and protein serine/threonine phosphatase 2A (PP2A) can all act as ERK2 phosphatases (Zhou et al, 2002a). Although ERK2 activity can be terminated by Tyr 185 dephosphorylation with high efficiency and fidelity by HePTP (Huang et al, 2004), PP2A regulates a wide variety of cellular signal transduction pathways, including the Raf1-MEK1/2-ERK1/2 signaling pathway at multiple steps, and can act as a negative or positive regulator of ERK1/2 activity (Zhou et al, 2002a;Ory et al, 2003).…”

Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3

“…Finally, it is important to note that interactions between the docking site and the KIM sequence alone may be only partially responsible for recognition of effector molecules by the MAP kinases (12,16). The role of the KIM͞docking site interaction is to increase the ''effective concentration'' of the interacting molecules (21,32). In addition to this ''tethering'' effect, additional interactions between the MAP kinases and their regulators and substrates (e.g., phosphorylation lip with MAP kinase͞ERK kinases and MKPs, and substrate binding region with the phosphorylation sequence in the substrates) are required to confer the exquisite specificity in MAP kinase signaling (16).…”