Molecular determinants of substrate recognition in hematopoietic protein-tyrosine phosphatase

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.     

The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.     

A kinetic approach for the study of protein phosphatase-catalyzed regulation of protein kinase activity

The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.     

Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site

Hematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.     

Lipid raft targeting of hematopoietic protein tyrosine phosphatase by protein kinase C theta-mediated phosphorylation

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.     

An essential role for mitogen-activated protein kinases, ERKs, in preventing heat-induced cell death.

Stimulation of mitogen-activated protein kinases (MAPKs) or extracellular signal regulated protein kinases (ERKs) after exposure of mammalian cells to ultraviolet (UV) and X-irradiation occurs through activation of receptor tyrosine kinases via Ras/Raf/Mek/ERKs cascade. This activation of MAPKs is proposed to play a role in the replacement of damaged proteins during these stresses. Heat shock also activates MAPKs; however, the signaling cascade and the biochemical and physiological links between activation by heat and downstream effects are unknown. In this report we demonstrate that, unlike irradiation, heat induces MAPKs through ceramide metabolism to sphingosine with stimulation of Raf-1 protein kinase. The activation of MAPKs by heat does not occur in all cell types, because the step(s) downstream of ceramide to activation of Raf-1 protein kinase is missing in myeloid leukemic cells such as HL-60, U937, and K562, while it is present in NIH3T3 fibroblasts. Heat-induced MAPK activation may enhance the ability of cells to survive a severe heat shock. Blocking 60-70% of the activity of MAPK (ERK1) by stable overexpression of the dominant negative allele ERK1-KR renders NIH3T3 and K562 cells up to 100-fold more sensitive to cytotoxic effects of heat. Conversely, NIH3T3 and K562 cells stably overexpressing the wild-type ERK1 develop resistance to killing by heat. These results suggest that increased thermal sensitivity of leukemic cells to thermal stress or other cancer therapy regimens could be attributable to lack of pertinent activation of the MAPK pathway by such stresses.     

Functions of MAP kinases: insights from gene-targeting studies

Mitogen-activated protein kinases (MAPKs) comprise a family of well-conserved serine/threonine kinases that control a vast array of physiological functions in a number of organisms ranging from yeast to mammals. Recently gene-targeting experiments have shed light on in vivo functions of MAPKs. In particular, embryos deficient in extracellular signal-regulated kinase (ERK) 2 lack mesoderm differentiation and placental angiogenesis. Knockout mice for c-Jun amino-terminal kinases have revealed roles for these kinases in neural apoptosis and activation/differentiation of T cells. Deletion of p38alpha MAPK results in angiogenic defects in the placenta and peripheral vessels. ERK5-deficient embryos are embryonic lethal due to defects in angiogenesis and cardiovascular development. Although these results have provided new insights for MAPK research, development and analysis of conditional knockout mice are required in order to investigate roles of MAPKs, especially, in other biological processes such as disease pathogenesis.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C sequence specific backbone assignment of the vanadate inhibited hematopoietic tyrosine phosphatase

The sequence-specific backbone assignment of hematopoietic protein tyrosine phosphatase (HePTP; PTPN7) in presence of vanadate has been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of HePTP in the presence of inhibitors to target leukemia and provide further insights into the function of protein tyrosine phosphatases.     

Hematopoietic Protein Tyrosine Phosphatase Mediates β2-Adrenergic Receptor-Induced Regulation of p38 Mitogen-Activated Protein Kinase in B Lymphocytes

Stimulation of the β₂-adrenergic receptor (β₂AR) on a CD40L/interleukin-4-activated B lymphocyte increases the level of immunoglobulin E (IgE) in a protein kinase A (PKA)- and p38 mitogen-activated protein kinase (MAPK)-dependent manner. However, the mechanism by which β₂AR stimulation mediates the increase in the level of p38 MAPK activation has remained unclear. Here we show that the β₂AR-induced increase in p38 MAPK activation occurred via a hematopoietic protein tyrosine phosphatase (HePTP)-mediated cross talk between PKA and p38 MAPK. β₂AR agonists, cAMP-elevating agents, and PKA inhibitors were used to show that β₂AR stimulation resulted in a PKA-dependent increase in p38 MAPK phosphorylation. Pharmacological agents and gene-deficient mice revealed that p38 MAPK phosphorylation was regulated by the G-stimulatory (Gs)/cAMP/PKA pathway independently of the G-inhibitory or β-arrestin-2 pathways. Coimmunoprecipitation and Western blot analysis showed that HePTP was phosphorylated in a PKA-dependent manner, which inactivated HePTP and allowed for increased free p38 MAPK to be phosphorylated by the MAPK cascade that was activated by CD40L. HePTP short hairpin RNA confirmed that HePTP played a role in regulating the level of p38 MAPK phosphorylation in a B cell. Thus, β₂AR stimulation on a B cell phosphorylates and inactivates HePTP in a Gs/cAMP/PKA-dependent manner to release bound p38 MAPK, making more available for phosphorylation and subsequent IgE regulation.     

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells.

This study was conducted on human Jurkat T cell lines to elucidate the role of EPA and DHA, n-3 PUFA, in the modulation of two mitogen-activated protein (MAP) kinases, that is, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). The n-3 PUFA alone failed to induce phosphorylation of ERK1/ERK2. We stimulated the MAP kinase pathway with anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA), which act upstream of the MAP kinase (MAPK)/ERK kinase (MEK) as U0126, an MEK inhibitor, abolished the actions of these two agents on MAP kinase activation. EPA and DHA diminished the PMA- and anti-CD3-induced phosphorylation of ERK1/ERK2 in Jurkat T cells. In the present study, PMA acts mainly via protein kinase C (PKC) whereas anti-CD3 antibodies act via PKC-dependent and -independent mechanisms. Furthermore, DHA and EPA inhibited PMA-stimulated PKC enzyme activity. EPA and DHA also significantly curtailed PMA- and ionomycin-stimulated T cell blastogenesis. Together these results suggest that EPA and DHA modulate ERK1/ERK2 activation upstream of MEK via PKC-dependent and -independent pathways and that these actions may be implicated in n-3 PUFA-induced immunosuppression.     

Cycloheximide-induced cPLA(2) activation is via the MKP-1 down-regulation and ERK activation

Extracellular signal-regulated kinase (ERK)-dependent phosphorylation is an important regulator for cytosolic phospholipase A(2) (cPLA(2)). In this study, we found that the protein synthesis inhibitor cycloheximide can potentiate thapsigargin-induced arachidonic acid (AA) release concomitant with ERK phosphorylation from murine RAW 264.7 macrophages. The cycloheximide effect is not due to the activation of p38 mitogen-activated protein kinase (MAPK) nor c-Jun NH(2)-terminal kinase (JNK), because the activator of both MAPKs anisomycin does not elicit AA release. Cycloheximide effect is additive to the tyrosine phosphatase inhibitor orthovanadate since these two stimuli induced sustained ERK activation respectively through inhibition of the translation and activity of MAPK phosphatase-1 (MKP-1).     

Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.     

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.