MKP-7, a novel mitogen-activated protein kinase phosphatase, functions as a shuttle protein

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) negatively regulate MAPK activity. In the present study, we have identified a novel MKP, designated MKP-7, and mapped it to human chromosome 12p12. MKP-7 possesses a long C-terminal stretch containing both a nuclear export signal and a nuclear localization signal, in addition to the rhodanese-like domain and the dual specificity phosphatase catalytic domain, both of which are conserved among MKP family members. When expressed in mammalian cells MKP-7 protein was localized exclusively in the cytoplasm, but this localization became exclusively nuclear following leptomycin B treatment or introduction of a mutation in the nuclear export signal. These findings indicate that MKP-7 is the first identified leptomycin B-sensitive shuttle MKP. Forced expression of MKP-7 suppressed activation of MAPKs in COS-7 cells in the order of selectivity, JNK p38 > ERK. Furthermore, a mutant form MKP-7 functioned as a dominant negative particularly against the dephosphorylation of JNK, suggesting that MKP-7 works as a JNK-specific phosphatase in vivo. Co-immunoprecipitation experiments and histological analysis suggested that MKP-7 determines the localization of MAPKs in the cytoplasm.     

Mitogen-activated protein kinase phosphatase (MKP)-1 in immunology, physiology, and disease

Mitogen-activated protein kinases (MAPKs) are key regulators of cellular physiology and immune responses, and abnormalities in MAPKs are implicated in many diseases. MAPKs are activated by MAPK kinases through phosphorylation of the threonine and tyrosine residues in the conserved Thr-Xaa-Tyr domain, where Xaa represents amino acid residues characteristic of distinct MAPK subfamilies. Since MAPKs play a crucial role in a variety of cellular processes, a delicate regulatory network has evolved to control their activities. Over the past two decades, a group of dual specificity MAPK phosphatases (MKPs) has been identified that deactivates MAPKs. Since MAPKs can enhance MKP activities, MKPs are considered as an important feedback control mechanism that limits the MAPK cascades. This review outlines the role of MKP-1, a prototypical MKP family member, in physiology and disease. We will first discuss the basic biochemistry and regulation of MKP-1. Next, we will present the current consensus on the immunological and physiological functions of MKP-1 in infectious, inflammatory, metabolic, and nervous system diseases as revealed by studies using animal models. We will also discuss the emerging evidence implicating MKP-1 in human disorders. Finally, we will conclude with a discussion of the potential for pharmacomodulation of MKP-1 expression.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions

MAP kinases (MAPKs) form a complex with MAPK kinases (MAPKKs), MAPK-specific phosphatases (MKPs) and various targets including MAPKAPKs. These docking interactions contribute to regulation of the specificity and efficiency of the enzymatic reactions. We have previously identified a docking site on MAPKs, termed the CD (common docking) domain, which is utilized commonly for docking interactions with MAPKKs, MKPs and MAPKAPKs. However, the CD domain alone does not determine the docking specificity. Here we have identified a novel site on p38 and ERK2 MAPKs that regulates the docking specificity towards MAPKAPKs. Remarkably, exchange of two amino acids in this site of ERK2 for corresponding residues of p38 converted the docking specificity for MAPKAPK-3/3pk, which is a dominant target of p38, from the ERK2 type to the p38 type, and vice versa. Furthermore, our detailed analyses with a number of MAPKAPKs and MKPs suggest that a groove in the steric structure of MAPKs, which comprises the CD domain and the site identified here, serves as a common docking region for various MAPK-interacting molecules.     

促分裂原活化蛋白激酶磷酸酶

促分裂原活化蛋白激酶磷酸酶（mitogen-activated protein kinase phosphatases,MKPs）是一类丝／苏氨酸和酪氨酸双特异性的磷酸酶。它在细胞分化、增殖和基因表达过程中起着重要的作用。MKPs可以选择性地结合促分裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）,对MAPK进行去磷酸化,从而调节MAPK信号通路的活性。另一方面,MAPK也可以激活MKPs,它们的相互作用确保了细胞内信号的精确传递,并参与细胞功能的调节。     

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.     

Solution structure of ERK2 binding domain of MAPK phosphatase MKP-3: structural insights into MKP-3 activation by ERK2

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.     

Modular structure of a docking surface on MAPK phosphatases

Mitogen-activated protein kinases (MAPKs) must be precisely inactivated to achieve proper functions in the cells. Ten members of dual specificity phosphatases specifically acting on MAPKs, termed MAPK phosphatases (MKPs), have been reported. Each member has its own substrate specificity that should be tightly regulated. However, the molecular mechanism underlying the regulation of the specificity is largely unknown. In the MAPK signaling pathways, docking interactions, which are different from transient enzyme-substrate interaction, are known to regulate the enzymatic specificity. Here we have identified and characterized a docking surface of MKPs. Our results show that a docking surface is composed of a tandem alignment of three subregions (modules): a cluster of positively charged amino acids, a cluster of hydrophobic amino acids, and a cluster of positively charged amino acids (positive-hydrophobic-positive). This modular structure well fits the docking groove on MAPKs that we have previously identified and may contribute to regulating the docking specificity of the MKP family. The position, number, and species of charged amino acids in each module including the central hydrophobic subregion are important factors in regulation of docking to specific MAPKs. This modular structure in the docking interaction may define a novel model of protein-protein interaction that would also regulate other systems.     

MKP-1在结核分枝杆菌感染中的作用和研究进展

有丝分裂原激活的蛋白激酶(Mitogen-Activated Protein Kinase,MAPK)信号通路是细胞感知外源性刺激并作出有效免疫应答的最重要的细胞内信号通路之一。近年来的研究表明:MAPK的表达异常与结核病的发生、发展密切相关。MAPK磷酸酶(MAPK phosphatases,MKPs)是一类在细胞内水解MAPKs家族的磷酸酶,通过负向调控MAPKs的活性,从而在调节细胞的应激、分化、增殖、凋亡等过程中发挥重要的作用,其中MKP-1是MKPs家族中被报道最多的成员,具有最强的去磷酸化能力。本文综述了MKP-1在结核分枝杆菌感染中的作用和研究进展。     

Expression, purification, and enzymatic characterization of the dual specificity mitogen-activated protein kinase phosphatase, MKP-4

Mitogen-activated protein kinase phosphatase-4 (MKP-4) is a dual specificity phosphatase, which acts as a negative regulator of insulin-stimulated pathways. Here, we describe expression, purification, and biochemical characterization of MKP-4. We used the Baculovirus expression system and purification with a combination of affinity and gel filtration chromatography to generate pure MKP-4 and MKP-4/p38 complex. Both MKP-4 and the MKP-4/p38 complex exhibited moderate activity toward the surrogate substrates p-nitrophenyl phosphate, 6, 8-difluoro-4-methylumbelliferyl phosphate, and 3-O-methylfluorescein phosphate. The phosphatase activity could be inhibited by peroxovanate, a potent inhibitor of protein tyrosine phosphatases. We further determined kinetic parameters for the MKP-4 and the MKP-4/p38 by using spectrophotometric and fluorescence intensity methods. The MKP-4/p38 complex was found to provide substantially higher phosphatase activity than MKP-4 alone, similar to what has been shown for MKP-3. Our data allow the configuration of screens for modulators of MKP-4 activity.     

Mice lacking MAP kinase phosphatase-1 have enhanced MAP kinase activity and resistance to diet-induced obesity

The mitogen-activated protein kinases (MAPK) play critical roles in the pathogenesis of diabetes and obesity. The MAPKs are inactivated by MAPK phosphatases (MKPs) either in the cytosol or nucleus. Here we show that mice lacking the nuclear-localized MKP, MKP-1 (mkp-1(-/-)), have enhanced Erk, p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) activities in insulin-responsive tissues as compared with wild-type mice. Although JNK promotes insulin resistance, mkp-1(-/-) mice exhibited unimpaired insulin-mediated signaling and glucose homeostasis. We reconciled these results by demonstrating that in mkp-1(-/-) mice, JNK activity was increased in the nucleus, but not the cytosol. Significantly, mkp-1(-/-) mice are resistant to diet-induced obesity due to enhanced energy expenditure, but succumb to glucose intolerance on a high fat diet. These results suggest that nuclear regulation of the MAPKs by MKP-1 is essential for the management of metabolic homeostasis in a manner that is spatially uncoupled from the cytosolic actions of the MAPKs.     

CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection

The macrophage-expressed CD40 regulates immune responses to Leishmania major infection by reciprocal signaling through p38 MAPK and ERK1/2. CD40-induced IL-10 or IL-12 plays crucial roles in the promotion or protection from L. major infection, respectively. Because p38 MAPK and ERK1/2 are dephosphorylated by dual-specificity MAPK phosphatases (MKPs), we tested the role of CD40 in the regulation of MKPs in L. major infection. MKP-1 expression and activity increased whereas MKP-3 expression and activity decreased in virulent L. major-infected macrophages. CD40 differentially regulated the expression and activity of MKP-1 and MKP-3, which, in turn, reciprocally regulated CD40-induced p38 MAPK and ERK1/2 phosphorylation and effector functions in macrophages. Triptolide, an inhibitor of MKP-1 expression, and lentivirally expressed MKP-1 short hairpin RNA enhanced CD40-induced anti-leishmanial functions and significantly protected susceptible BALB/c mice from L. major infection. Similarly, lentivirally overexpressed MKP-3 significantly reduced disease progression and parasite burden in susceptible BALB/c mice. Thus, to our knowledge, our data show for the first time that CD40 reciprocally regulates MKP-1 and MKP-3 expression and activity while the MKPs contribute to the reciprocal CD40 signaling-regulated anti-leishmanial functions. The findings reveal a novel parasite-devised immune evasion strategy and an effective target to redirect CD40-regulated immune responses.     

The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38

Mitogen-activated protein (MAP) kinases are critical mediators of innate immune responses. In response to lipopolysaccharide (LPS), MAP kinases are rapidly activated and play an important role in the production of proinflammatory cytokines. Although a number of MAP kinase phosphatases (MKPs) have been identified, their roles in the control of cytokine production have not been well defined. In the present report, we investigated the role of MKP-1 in alveolar macrophages stimulated with LPS. We found that LPS triggered transient activation of three MAP kinase subfamilies, ERK, JNK, and p38, in both immortalized and primary murine alveolar macrophages. MKP-1 was rapidly induced by LPS, and its induction correlated with the dephosphorylation of these MAP kinases. Blocking MKP-1 with triptolide prolonged the activities of both JNK and p38 in immortalized alveolar macrophages. Stimulation of primary alveolar macrophages isolated from MKP-1-deficient mice with LPS resulted in a prolonged p38 phosphorylation compared with wild type alveolar macrophages. Accordingly, these MKP-1-deficient alveolar macrophages also mounted a more robust and rapid tumor necrosis factor alpha production than their wild type counterparts. Adenovirus-mediated MKP-1 overexpression significantly attenuated tumor necrosis factor alpha production in immortalized alveolar macrophages. Finally, MKP-1 was induced by a group of corticosteroids frequently prescribed for the treatment of inflammatory lung diseases, and the anti-inflammatory potencies of these drugs closely correlated with their abilities to induce MKP-1. Our studies indicated that MKP-1 plays an important role in dampening the inflammatory responses of alveolar macrophages. We speculate that MKP-1 may represent a novel target for therapeutic intervention of inflammatory lung diseases.     

MKP-2: out of the DUSP-bin and back into the limelight

The MKPs (mitogen-activated protein kinase phosphatases) are a family of at least ten DUSPs (dual-specificity phosphatases) which function to terminate the activity of the MAPKs (mitogen-activated protein kinases). Several members have already been demonstrated to have distinct roles in immune function, cancer, fetal development and metabolic disorders. One DUSP of renewed interest is the inducible nuclear phosphatase MKP-2, which dephosphorylates both ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) in vitro. Recently, the understanding of MKP-2 function has been advanced due to the development of mouse knockout models, which has resulted in the discovery of novel roles for MKP-2 in the regulation of sepsis, infection and cell-cycle progression that are distinct from those of other DUSPs. However, many functions for MKP-2 still await to be characterized.