Mitogen-activated protein kinase phosphatase-1 (MKP-1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes

We recently reported a diurnal and norepinephrine (NE) -induced expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the rat pineal gland and postulated that this MKP-1 expression might impact adrenergic-regulated arylalkylamine- N -acetyltransferase (AA-NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP-1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP-1 in other cell types and small interfering RNA targeted against Mkp1 in NE-stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP-1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA-NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE-stimulated AA-NAT activity. Interestingly, suppressing MKP-1 expression had no effect on the time profile of NE-stimulated p38MAPK activation. These results indicate that MKP-1 modulates the profile of AA-NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.     

MKP—1在血管紧张素Ⅱ导致心肌肥大反应中的调控作用

本研究主要从丝裂原活化蛋白激酶磷酸酶－１（ＭＫＰ－１）角度,研究丝裂原活化蛋白激酶（ＭＡＰＫ）信号途径在血管紧张素Ⅱ介导的新生大鼠心肌细胞肥大反应中的作用及调控机制。实验以心肌细胞蛋白合成速率、蛋白含量及细胞表面积作为心肌肥大反应的指标,以凝胶内ＭＢＰ原位磷酸化测定ＭＡＰＫ活性,以免疫印迹法（Ｗｅｓｔｅｒｎ ｂｏｌｔｔｉｎｇ）分别测定ＭＫＰ－１及磷酸化ｐ４４ＭＡＰＫ、ｐ４２ＭＡＰＫ蛋白表达。结果发     

Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.     

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.     

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.     

17beta -Estradiol modulates mechanical strain-induced MAPK activation in mesangial cells.

Gender is an important determinant of clinical outcome across a broad spectrum of kidney diseases, but the mechanism(s) responsible for the protective effect of female gender have not been fully elucidated. Remnant kidney glomerular injury is limited in female rats compared with male rats despite similar elevations in glomerular capillary pressure. In vitro, mechanical strain leads to the activation of p44/42 mitogen-activated kinase (p44/42 MAPK) and Jun N-terminal kinase/stress-activated protein kinase (SAPK) in glomerular mesangial cells (MC). Accordingly, we studied the effect of 17beta-estradiol on mechanical strain-induced signal transduction in MC. Exposure of MC to mechanical strain increased p44/42 MAPK activation (3-fold) and SAPK activation (2.5-fold), and kinase activation was inhibited by pretreatment with 17beta-estradiol (10(minus sign8) to 10(minus sign11) m) for 24 h in a dose-dependent manner. Mechanical strain-induced nuclear translocation of p44/42 MAPK and SAPK and nuclear protein binding to AP-1 were also attenuated by 17beta-estradiol. The inhibitory effects of 17beta-estradiol were not reproduced by the cell-impermeable estrogen, BSA/17beta-estradiol, nor did preincubation with 17beta-estradiol lead to actin cytoskeleton disassembly or impaired stress fiber formation. However, 17beta-estradiol did increase base-line levels of the dual specificity phosphatase MKP-1. The inhibitory effects of 17beta-estradiol on p44/42 MAPK activation and SAPK activation, translocation, and AP-1 binding were all abrogated by the estrogen receptor antagonist, ICI-182,780. We conclude that attenuation of mechanical strain-induced MAPK activation by 17beta-estradiol is dependent on intracellular estrogen receptor. The attenuation of stretch-induced kinase activation may be due, at least in part, to an effect of 17beta-estradiol on MKP-1 expression. Together, these findings add insight into the protective effect of gender on renal disease progression.     

CaMKII protects MKP-1 from proteasome degradation in endothelial cells

CaMKs are a widely distributed family of kinases with multiple and often cell specific effects on intracellular signal transduction pathway. In endothelial cells, it has been recognized a role for CamKII in several pathways such as eNOS activation and nitric oxide production. It is not clear though, whether CaMKII interfere with other endothelial cell functions such as ERK activation and cell proliferation. We explored this issue in primary cultured rat endothelial cells and we evaluated the effect on endothelial cell proliferation and DNA synthesis. CaMKII inhibition through Cantide, conducted into the cell through Antoennapedia (ANT-CN), showed positive effects on proliferation and H³-thimdine incorporation similar to insulin stimulation. Accordingly, both CaMKII pharmacological inhibition and silencing through shRNA produced activation of the p44/42 MAPK. These observations leaded to the hypothesis that CamKII could regulate p44/p42 by interfering with specific ERK phosphatases. Indeed, we found that CaMKII interacts and protect the dual specific phosphatase MKP-1 from proteasome mediated degradation while this complex is disrupted by CaMKII inhibitors. This study reveals that CaMKII, besides phosphorylation through the known ras-raf-mek pathway, can regulate also dephosphorylation of p44/p42 by modulation of MKP-1 level. This novel finding opens to a novel scenario in regulation of endothelial cell functions.     

MicroRNA-101 targets MAPK phosphatase-1 to regulate the activation of MAPKs in macrophages

MAPK phosphatase-1 (MKP-1) is an archetypical member of the dual-specificity phosphatase family that deactivates MAPKs. Induction of MKP-1 has been implicated in attenuating the LPS- or peptidoglycan-induced biosynthesis of proinflammatory cytokines, but the role of noncoding RNA in the expression of the MKP-1 is still poorly understood. In this study, we show that MKP-1 is a direct target of microRNA-101 (miR-101). Transfection of miR-101 attenuates induction of MKP-1 by LPS as well as prolonged activation of p38 and JNK/stress-activated protein kinase, whereas inhibition of miR-101 enhances the expression of MKP-1 and shortens p38 and JNK activation. We also found that expression of miR-101 is induced by multiple TLR ligands, including LPS, peptidoglycan, or polyinosinic-polycytidylic acid, and that inhibition of PI3K/Akt by LY294002 or Akt RNA interference blocks the induction of miR-101 by LPS in RAW264.7 macrophage cells. Moreover, treatment of cells with dexamethasone, a widely used anti-inflammatory agent, markedly inhibits miR-101 expression and enhances the expression of MKP-1 in LPS-stimulated macrophages. Together, these results indicate that miR-101 regulates the innate immune responses of macrophages to LPS through targeting MKP-1.     

Signaling through the p38 and p42/44 mitogen-activated families of protein kinases in pancreatic beta-cell proliferation

The present study has focused on the role of the 42- and 44-kDa mitogen-activated protein kinases (p42/44 MAPKs) and the 38-kDa mitogen-activated protein kinase (p38 MAPK) in the proliferation of the pancreatic beta-cell line MIN6. MIN6 beta-cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Inhibition of both the p42/44 MAPK pathway using the MEK inhibitor PD098059 (PD) and the p38 MAPK pathway using the p38 inhibitor SB203580 (SB) caused a marked, concentration-dependent reduction in the BrdU immunostaining observed in the presence of 15% FCS when assessed using fluorescence immunocytochemistry. These data provide direct evidence of a role for p42/44 MAPKs in the mitogenic response of MIN6 beta-cells to FCS. Furthermore, these data also suggest a novel role for the p38 MAPK pathway in MIN6 beta-cell proliferation.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

Ethanol modulates the growth of human breast cancer cells in vitro

The role of ethanol or its metabolites on breast neoplasm has not been characterized. We hypothesized that ethanol may alter the growth rate of human breast tumor epithelial cells by modulating putative growth-promoting signaling pathways such as p44/42 mitogen-activated protein kinases (MAPKs). The MCF-7 cell line, considered a suitable model, was used in these studies to investigate the effects of ethanol on [(3)H]thymidine incorporation, cell number, and p44/42 MAPK activities in the presence or absence of a MAPK or extracellular signal-regulated kinase ERK-1, and (MEK1) inhibitor (PD098059). Treatment of MCF-7 cells with a physiologically relevant concentration of ethanol (0.3% or 65 mM) increased p44/42 activities by an average of 400% (P < 0.02), and subsequent cell growth by 200% (P < 0.05) in a MEK1 inhibitor (PD098059)-sensitive fashion, thus suggesting that the Ras/MEK/MAPK signaling pathways are crucial for ethanol-induced MCF-7 cell growth.     

Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling

The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339-342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser(296)/Ser(323) domain, which was not affected by substituting Ala for Ser at Ser(359)/Ser(364). The DEF and Ser(296)/Ser(323) sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser(359)/Ser(364) sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser(296) MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser(296) MKP-1 bound to SCF(Skp2) ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCF(Skp2) cooperation, thereby sustaining ERK activation.     

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.     

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。