Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.
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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. (+info)
Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.
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Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor. (+info)
MKP5, a new member of the MAP kinase phosphatase family, which selectively dephosphorylates stress-activated kinases.
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Dual-specificity protein tyrosine phosphatases are a burgeoning family of enzymes, some of which, the MKPs, are implicated in the regulation of mitogen-activated protein (MAP) kinases. MKPs have been shown to reverse the activation of the MAP kinases by hydrolyzing phosphothreonine and phosphotyrosine residues present in the substrates. Here we describe the characterization of a novel member of the MKP family, MKP5. The MKP5 gene, which maps to human chromosome 1q32, is expressed tissue-specifically as two transcripts of approximately 3.4 and 2.4 kb in human liver and skeletal muscle. When expressed in mammalian cells, MKP5 blocks the enzymatic activation of MAP kinases with the selectivity p38 approximately JNK/SAPK >> ERK. Immunoprecipitation of endogenous MAP kinases by the catalytically inactive transfected MKP5 demonstrates that it preferentially binds to the p38 and JNK/SAPK kinases. These findings suggest that the selectivity of this phosphatase may be determined at least in part at the level of substrate binding. (+info)
Identification and characterization of genes upregulated in cells transformed by v-Jun.
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The transcription factor Jun (c-Jun) functions as a recipient of extracellular growth signals and converts them into patterns of gene expression. An oncogenic variant of c-Jun was isolated from the acutely transforming retrovirus ASV17. Overexpression of this viral Jun (v-Jun) induces transformation of chicken embryo fibroblasts (CEF) in culture and fibrosarcomas in chickens. v-Jun is a constitutively active form of c-Jun and transforms cells presumably by deregulating the expression of specific target genes. In this report, we describe six genes whose transcripts are upregulated in v-Jun-transformed CEF. Three of these genes show homology to known mammalian genes, to MAP kinase phosphatase 2 (MKP-2), to reversion-induced LIM protein (RIL) and to cytokine-inducible SH2-containing protein (CIS). Northern blot analysis, using CEF infected with various Jun mutants or an estrogen-regulatable Jun chimera, revealed distinct induction patterns of individual targets by v-Jun. The chicken RIL homolog showed an expression pattern tightly correlated with the activity of v-Jun. Its expression is also transformation-dependent, suggesting a role for this gene in v-Jun transformation. The newly identified v-Jun targets can serve as molecular markers in the v-Jun transformation process. Oncogene (2000) 19, 3537 - 3545 (+info)
Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6.
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TCR and CD28 costimulatory receptor-cooperative induction of T cell IL-2 secretion is dependent upon activation of mitogen-activated protein (MAP) kinases. Using yeast-hybrid technology, we cloned a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, MAP kinase phosphatase-6 (MKP6), which we demonstrate inactivates MAP kinases. Several lines of evidence indicate that MKP6 plays an important functional role in CD28 costimulatory signaling. First, in human peripheral blood T cells (PBT), expression of MKP6 is strongly up-regulated by CD28 costimulation. Second, transfer of dominant-negative MKP6 to PBT with the use of retroviruses primes PBT for the secretion of substantially larger quantities of IL-2, specifically in response to CD28 costimulation. A similar enhancement of IL-2 secretion is observed neither in response to TCR plus CD2 costimulatory receptor engagement nor in response to other mitogenic stimuli such as phorbol ester and ionomycin. Furthermore, this hypersensitivity to CD28 costimulation is associated with CD28-mediated hyperactivation of MAP kinases. Third, a retroviral transduced chimeric receptor with a CD28 CYT that is specifically unable to bind MKP6 costimulates considerably larger quantities of IL-2 from PBT than a similar transduced chimeric receptor that contains a wild-type CD28 CYT. Taken together, these results suggest that MKP6 functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls the activation of MAP kinases. (+info)
Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.
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The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress. (+info)
A Novel MAPK phosphatase MKP-7 acts preferentially on JNK/SAPK and p38 alpha and beta MAPKs.
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Mitogen-activated protein kinases (MAPKs) are inactivated via dephosphorylation of either the threonine or tyrosine residue or both in the P-loop catalyzed by protein phosphatases which include serine/threonine phosphatases, tyrosine phosphatases, and dual specificity phosphatases. Nine members of the dual specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has its own substrate specificity, tissue distribution, and subcellular localization. In this study, we have cloned and characterized a novel MKP, designated MKP-7. MKP-7 is most similar to hVH5, a member of previously known MKPs, in the primary structure. MKP-7 is predominantly localized in the cytoplasm when expressed in cultured cells, whereas hVH5 is both in the nucleus and the cytoplasm. MKP-7 binds to and inactivates p38 MAPK and JNK/SAPK, but not ERK. Furthermore, we have found that MKPs have the substrate specificity toward the isoforms of the p38 family (alpha, beta, gamma, and delta). MKP-7 binds to and inactivates p38 alpha and -beta, but not gamma or delta. MKP-5 and CL100/MKP-1 also bind to p38 alpha and -beta, but not gamma or delta. Finally, we propose a tentative classification of MKPs based on the sequence characteristics of their MAPK-docking site. (+info)
Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MAP kinase phosphatase-2 and their ability to activate the phosphatase catalytically.
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MKP-2 is a member of the mitogen-activated protein (MAP) kinase phosphatase family which has been suggested to play an important role in the feedback control of MAP kinase-mediated gene expression. Although MKP-2 preferentially inactivates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) MAP kinase subfamilies, the mechanisms underlying its own regulation remain unclear. In this report, we have examined the MKP-2 interaction with and catalytic activation by distinct MAP kinase subfamilies. We found that the catalytic activity of MKP-2 was enhanced dramatically by ERK and JNK but was affected only minimally by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, whereas JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the NH(2)-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised the catalytic activation of MKP-2 by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 whereas MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for the substrate preference of MKP-2 and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function. (+info)