Extracellular signal-regulated kinases in T cells: characterization of human ERK1 and ERK2 cDNAs.

Extracellular signal-regulated kinases 1 and 2 are growth factor-sensitive serine/threonine kinases. cDNAs for both human kinases were isolated and sequenced. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 1 were 88% and 96% identical, respectively, to the homologous rat sequences. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 2 were 90% and 98% identical, respectively, to the corresponding rat sequences. A human extracellular signal-regulated kinase 2 specific probe was used to demonstrate that the mRNA for this kinase was present in T cells and did not change with activation. The deduced protein sequences of both human kinases were greater than 95% identical to two Xenopus kinase sequences, indicating that these enzymes are highly conserved across species.     

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

Role for DUSP1 (dual-specificity protein phosphatase 1) in the regulation of autophagy

Accumulating evidence suggests that mitogen-activated protein kinases (MAPKs) regulate macroautophagy/autophagy. However, the involvement of dual-specificity protein phosphatases (DUSPs), endogenous inhibitors for MAPKs, in autophagy remains to be determined. Here we report that DUSP1/MKP-1, the founding member of the DUSP family, plays a critical role in regulating autophagy. Specifically, we demonstrate that DUSP1 knockdown by shRNA in human ovarian cancer CAOV3 cells and knockout in murine embryonic fibroblasts, increases both basal and rapamycin-increased autophagic flux. Overexpression of DUSP1 had the opposite effect. Importantly, knockout of Dusp1 promoted phosphorylation of ULK1 at Ser555, and BECN1/Beclin 1 at Ser15, and the association of PIK3C3/VPS34, ATG14, BECN1 and MAPK, leading to the activation of the autophagosome-initiating class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Furthermore, knockdown and pharmacological inhibitor studies indicated that DUSP1-mediated suppression of autophagy reflected inactivation of the MAPK1-MAPK3 members of the MAPK family. Knockdown of DUSP1 sensitized CAOV3 cells to rapamycin-induced antigrowth activity. Moreover, CAOV3-CR cells, a line that had acquired cisplatin resistance, exhibited an elevated DUSP1 level and were refractory to rapamycin-induced autophagy and cytostatic effects. Knockdown of DUSP1 in CAOV3-CR cells restored sensitivity to rapamycin. Collectively, this work identifies a previously unrecognized role for DUSP1 in regulating autophagy and suggests that suppression of DUSP1 may enhance the therapeutic activity of rapamycin.     

Mitochondrial extracellular signal-regulated kinases 1/2 (ERK1/2) are modulated during brain development

Intracellular activation and trafficking of extracellular signal-regulated protein kinases (ERK) play a significant role in cell cycle progression, contributing to developmental brain activities. Additionally, mitochondria participate in cell signalling through energy-linked functions, redox metabolism and activation of pro- or anti-apoptotic proteins. The purpose of the present study was to analyze the presence of ERK1/2 in mitochondria during rat brain development. Immunoblotting, immune electron microscopy and activity assays demonstrated that ERK1/2 are present in fully active brain mitochondria at the outer membrane/intermembrane space fraction. Besides, it was observed that ERK1/2 translocation to brain mitochondria follows a developmental pattern which is maximal between E19-P2 stages and afterwards declines at P3, just before maximal translocation to nucleus, and up to adulthood. Most of mitochondrial ERK1/2 were active; upstream phospho-MAPK/ERK kinases (MEK1/2) were also detected in the brain organelles. Mitochondrial phospho-ERK1/2 increased at 1 microm hydrogen peroxide (H(2)O(2)) concentration, but it decreased at higher 50-100 microm H(2)O(2), almost disappearing after the organelles were maximally stimulated to produce H(2)O(2) with antimycin. Our results suggest that developmental mitochondrial activation of ERK1/2 cascade contributes to its nuclear translocation effects, providing information about mitochondrial energetic and redox status to the proliferating/differentiating nuclear pathways.     

Extracellular signal-regulated kinases (ERK) 1, 2 are required for luteinizing hormone (LH)-induced steroidogenesis in primary Leydig cells and control steroidogenic acute regulatory (StAR) expression

The luteinizing hormone (LH) plays a critical role in steroidogenesis, by stimulating cAMP-dependent protein kinase A (PKA) and phospholipase A2 activity, and by mobilizing calcium and chloride ions. In contrast, whether the ERK 1, 2 mitogen-activated protein (MAP) kinases are involved in LH-induced steroidogenesis is less obvious. Here, we sought to clarify this point in rat primary Leydig cells, naturally bearing the LH receptor (LH-R) in male, and in the mouse tumoral Leydig cell line (MLTC 1). Pre-incubation of both cell types with the mitogen-activated protein kinase kinase (MEK) inhibitors U0126 and PD98059 reduced LH-induced steroidogenesis, and tonically enhanced the expression of the StAR protein. Furthermore, ERK1, 2 were inducibly phosphorylated following LH exposure of MLTC 1 cells. Altogether, our results indicate that in primary as well as in tumoral Leydig cells, inhibiting MEK dampened LH-induced steroidogenesis but enhanced basal as well as LH-induced StAR expression, suggesting that ERK1,2 could be involved in these responses.     

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~ 5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~ 30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their k_cat values were estimated to be minimally ~ 30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.     

Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases.

Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of protein-tyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.     

Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location.     

The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria

Bcl-2 protein play important roles in the regulation of apoptosis. We previously reported that the phosphorylation of Bcl-2 was augmented by treatment with protein phosphatase 2A (PP2A) inhibitor; however, the kinase responsible for Bcl-2 phosphorylation had not yet been identified. In this study, we identified extracellular-signal-regulated kinase (ERK) as the responsible kinase for the phosphorylation of Bcl-2. We also found that the transmembrane region (TM) deleted form of Bcl-2 (Bcl-2DeltaTM), which was unable to localize on the mitochondria was constitutively phosphorylated, whereas wild-type Bcl-2 that localized on the mitochondria, was present in its hypophosphorylated form. The phosphorylation of Bcl-2DeltaTM was retarded by treatment with MAP kinase ERK kinase (MEK) inhibitor and PP2A did not bind to Bcl-2DeltaTM. These observations suggest that Bcl-2DeltaTM is constitutively phosphorylated by ERK, but is not dephosphorylated by PP2A in human tumor cell lines. The phosphorylation of Bcl-2 resulted in a reduction in anti-apoptotic function, implying that dephosphorylation promoted the anti-apoptotic activity of Bcl-2 protein in human tumor cell lines. Thus, the present findings suggest that ERK and PP2A are physiological regulators of Bcl-2 phosphorylation, and these enzymes exert an influence on the anti-apoptotic function of Bcl-2.     

Distinct Roles for Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 in the Structure and Production of a Primate Gammaherpesvirus

During their progression from intranuclear capsids to mature trilaminar virions, herpesviruses incorporate an extensive array of viral as well as a smaller subset of cellular proteins. Our laboratory previously reported that rhesus monkey rhadinovirus (RRV), a close homolog of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is comprised of at least 33 different virally encoded proteins. In the current study, we found that RRV infection activated the extracellular signal-regulated kinase (ERK) pathway and nascent virions preferentially incorporated the activated form of ERK2 (pERK2) into the tegument. This was evident even in the face of greatly diminished stores of intracellular ERK2, suggesting a clear bias toward the incorporation of pERK2 into the RRV particle. Similar to earlier findings with KSHV, activation of ERK was essential for the production of lytic viral proteins and virions. Knockdown of intracellular ERK, however, failed to inhibit virus production, likely due to maintenance of residual pools of intracellular pERK2. Paradoxically, selective knockdown of ERK1 enhanced virion production nearly 5-fold and viral titers more than 10-fold. These data are the first to implicate ERK1 as a negative regulator of lytic replication in a herpesvirus and the first to demonstrate the incorporation of an activated signaling molecule within a herpesvirus. Together, the results further our understanding of how herpesviruses interact with host cells during infection and demonstrate how this family of viruses can exploit cellular signal transduction pathways to modulate their own replication.     

Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.     

Induction of dual-specificity phosphatase 1 (DUSP1) by pulsatile gonadotropin-releasing hormone stimulation: role for gonadotropin subunit expression in mouse pituitary LbetaT2 cells

The adherens junction (AJ) is important for maintaining uterine structural integrity, composition of the luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barriers. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin, and alpha-catenin at their mRNA and protein levels, together with the cellular distribution of meprinbeta, phospho-beta-catenin, and active beta-catenin proteins, in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation. By in situ hybridization and immunofluorescence, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. Immunofluorescence study showed no change in epithelial expression patterns of uterine AJ proteins from Days 1 to 5 of pregnancy. With advancement of the implantation process, AJ components were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in the PDZ, but not in the SDZ, of mice. Taken together, these results suggest that AJ proteins contribute to uterine barrier functions by cell-cell adhesion to ensure protection of the embryo. In addition, cleavage of E-cadherin by meprinbeta might contribute to weakening uterine epithelial cell-cell contact for blastocyst implantation. We also report that the nuclear localization of active beta-catenin from Day 4 onward in hamsters implies that beta-catenin/Wnt-signal transduction is activated in the uterus during implantation and decidualization.     

Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.     

Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease

The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT. In vivo, calcineurin mediates prolonged dephosphorylation of Slm1 and Slm2 during heat stress, and this response can be mimicked by exogenous addition of the sphingoid base phytosphingosine. Slm proteins also promote the growth of yeast cells in the presence of myriocin, an inhibitor of sphingolipid biosynthesis, and regulation of Slm proteins by calcineurin is required for their full activity under these conditions. During heat stress, sphingolipids signal turnover of the uracil permease, Fur4. In cells lacking Slm protein activity, stress-induced endocytosis of Fur4 is blocked, and Fur4 accumulates at the cell surface in a ubiquitinated form. Furthermore, cells expressing a version of Slm2 that cannot be dephosphorylated by calcineurin display an increased rate of Fur4 turnover during heat stress. Thus, calcineurin may modulate sphingolipid-dependent events through regulation of Slm1 and Slm2. These findings, in combination with previous work identifying Slm1 and Slm2 as targets of Mss4/phosphatidylinositol 4,5-bisphosphate and TORC2 signaling, suggest that Slm proteins integrate information from a variety of signaling pathways to coordinate the cellular response to heat stress.     

A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production.

Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia. Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities. Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types. Because the mitogen-activated protein kinases ERK-1 and ERK-2 have been implicated in the control of many immune responses, we tested the concept that they are a key indicator for differences in cellular LPS sensitivity. We observed that murine RAW 264.7 macrophages and murine BV-2 microglial cells both respond to LPS by exhibiting increased IkappaBalpha degradation, enhanced NF-kappaB DNA binding activity, and elevated nitric oxide and interleukin-1beta production. Although LPS potently stimulates ERK activation in RAW 264.7 macrophages, it does not activate ERK-1/-2 in BV-2 microglia. Moreover, antagonism of the MEK/ERK pathway potentiates LPS-stimulated nitric oxide production, suggesting that LPS-stimulated ERK activation can exert inhibitory effects in macrophage-like cells. These data support the idea that ERK activation is not a required function of LPS-mediated signaling events and illustrate that alternative/additional pathways for LPS action exist in these cell types.     

Requirement for protein-tyrosine phosphatase SHP-2 in insulin-induced activation of c-Jun NH(2)-terminal kinase

Mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinases (JNKs), are activated by insulin. Although the mechanism by which the insulin receptor activates extracellular signal-regulated kinases is relatively well defined, the pathway that leads to JNK activation is poorly understood. Overexpression of a catalytically inactive mutant (SHP-2C/S) of the protein-tyrosine phosphatase SHP-2 in Rat-1 fibroblasts that also express human insulin receptors has now revealed that activation of JNKs by insulin and epidermal growth factor, but not that by anisomycin or sorbitol, requires SHP-2. A dominant negative mutant (RasN17) of Ha-Ras blocked insulin-induced JNK activation, whereas a dominant negative mutant (RacN17) of Rac1 or a specific inhibitor (LY294002) of phosphoinositide 3-kinase did not, indicating a role for Ras, but not for Rac or phosphoinositide 3-kinase, in this effect. SHP-2C/S markedly inhibited Ras activation in response to insulin without affecting insulin-induced tyrosine phosphorylation of cellular substrates or the dissociation of the Crk-p130(Cas) complex. In contrast, SHP-2C/S did not inhibit activation of JNKs induced by a constitutively active mutant (RasV12) of Ha-Ras. Furthermore, expression of myristoylated SOS, which functions as a potent activator of Ras, induced JNK activation even when SHP-2 was inactivated. These results suggest that SHP-2 contributes to JNK activation in response to insulin by positively regulating the Ras signaling pathway at the same level as, or upstream from, SOS.