Transcriptional activation of dual specificity phosphatases: a feedback loop

Phorbol ester tumor promoters, such as phorbol 12-myristate 13-acetate (PMA), are potent activatorsof extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase (SAPK), and p38mitogen-activated protein kinase (MAPK) in U937 human leukemic cells. These kinases are regulatedby the reversible dual phosphorylation of conserved threonine and tyrosine residues. The dualspecificity protein phosphatase MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylateand inactivate ERK2, SAPK, and p38 MAPK in transient transfection studies. PMA treatment induces MKP-1 protein expression in U937 cells, which is detectable within 30min with maximal levels attained after 4 h. This time course coincides with the rapid inactivation ofPMA-induced SAPK activity, but not ERK2 phosphorylation, which remains elevated for up to 6 h. Toexamine directly the role of MKP-1 in the regulation of these protein kinases in vivo, aU937 cell line was established that conditionally expresses MKP-1 from the human metallothionein IIa promoter.Conditional expression of MKP-1 inhibits PMA-induced ERK2, SAPK, and p38 MAPK activity. However, bytitrating the levels of MKP-1 expression from the human metallothionein IIa promoter it wasfound that p38 MAPK and SAPK are much more sensitive to inhibition by MKP-1 than ERK2. Thisdifferential substrate specificity of MKP-1 can be functionally extended to nuclear transcriptionalevents because PMA-induced c-Jun transcriptional activity is more sensitive to inhibition by MKP-1than either Elk-1 or c-Myc. Conditional expression of MKP-1 also abolishes the induction ofendogenous MKP-1 protein expression in response to PMA treatment. This negative feedbackregulatory mechanism is likely due to MKP-1-mediated inhibition of ERK2, since studies utilizing theMEK1/2 inhibitor PD98059 suggest that ERK2 activation is required for PMA-induced MKP-1expression. These findings suggest that ERK2-mediated induction of MKP-1 may play an importantrole in preferentially attenuating signaling through the p38 MAPK and SAPK signal transductionpathways (Franklin, 1997).

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and MKP-2 are two members of arecently described family of dual specificity phosphatases that are capable of dephosphorylatingp42/p44MAPK. Overexpression of MKP-1 or MKP-2 inhibits MAP kinase-dependent intracellularsignaling events and fibroblast proliferation. MKP-1 and MKP-2 are not expressed in quiescentcells, but are rapidly induced following serum addition, with protein detectable as early as 30 min(MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 and MKP-2 is sustained, with proteindetectable up to 14 h after serum addition. Induction of MKP-1 and (to a lesser extent) MKP-2temporally correlates with p42/p44MAPK inactivation. To analyze the contribution of the MAP kinasecascade to MKP-1 and MKP-2 induction, CCL39 cells were examined that had been transformed with either v-ras ora constitutively active direct upstream activator of MAP kinase, mitogen-activated protein kinasekinase-1 (MKK-1; MKK-1[SD/SD] mutant). In both cell models, MKP-1 and MKP-2 areconstitutively expressed, with MKP-2 being prevalent. CCL39 cells expressing anestradiol-inducible deltaRaf-1::ER chimera, activation of Raf alone is sufficient to induce MKP-1 andMKP-2. The role of the MAP kinase cascade in MKP induction was highlighted by the MKK-1inhibitor PD 098059, which blunts both the activation of p42/p44MAPK and the induction of MKP-1and MKP-2. However, the MAP kinase cascade is not absolutely required for the induction of MKP-1,as this phosphatase, but not MKP-2, is induced to detectable levels by agents that stimulate proteinkinases A and C. Thus, activation of the p42/p44MAPK cascade promotes the induction of MKP-1and MKP-2, which may then attenuate p42/p44MAPK-dependent events in an inhibitory feedbackloop (Brondello, 1997).

Insulin signaling involves the transient activation/inactivation of various proteins by a cycle ofphosphorylation/dephosphorylation. This dynamic process is regulated by the action of protein kinasesand protein phosphatases. One family of protein kinases that is important in insulin signaling is themitogen-activated protein (MAP) kinases, whose action is reversed by specific MAP kinasephosphatases (MKPs). Insulin stimulation of Hirc B cells overexpressing the human insulin receptorresults in increased MKP-1 mRNA levels. MKP-1 mRNA increases in a dose-dependent manner toa maximum of 3- to 4-fold over basal levels within 30 min, followed by a gradual return to basal. ThemRNA induction does not require the continuous presence of insulin. The induction of MKP-1 proteinsynthesis follows MKP-1 mRNA induction; MKP-1 protein is maximally expressed after 120 minof insulin stimulation. MKP-1 mRNA induction by insulin requires insulin receptor tyrosine kinaseactivity, since overexpression of an altered insulin receptor with impaired intrinsic tyrosine kinaseactivity prevents mRNA induction. Forskolin, (Bu)2-cAMP, 8-bromo-cAMP, and8-(4-chlorophenylthio)-cAMP increase the MKP-1 mRNA content moderately above basal. Theseagents also augment the insulin-stimulated expression of MKP-1 mRNA. However, in some casesthe increase in MKP-1 mRNA expression is less than additive. Nevertheless, these results indicatethat multiple signaling motifs might regulate MKP-1 expression and suggest another mechanism for theattenuation of insulin-stimulated MAP kinase activity by cAMP. Overexpression of MKP-1 in Hirc Bcells inhibits both insulin-stimulated MAP kinase activity and MAP kinase-dependent genetranscription. The results of these studies lead to the conclusion that insulin regulates MKP-1 and stronglysuggest that MKP-1 acts as a negative regulator of insulin signaling (Kusari, 1997).

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results ina biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment ofRat-1 cells with either cycloheximide or sodium orthovanadate has little effect on the early peak ofERK1 activity but potentiates the sustained phase. Cycloheximide also potentiates ERK1 activation inRat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1.Since cycloheximide does not potentiate MEK activity but abrogates the expression of mitogen-activatedprotein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, it was speculated thatthe level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells.Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selectiveinhibitor of Gi-protein-coupled signaling pathways, reduces LPA-stimulated MKP-1 expression by only50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER has little or no effect onMKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but notsufficient for MKP-1 expression in Rat-1 cells. Activation of PKC plays little part in growthfactor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression is blocked bybuffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation withoutpotentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, a strong synergy of MKP-1expression is observed when cells are stimulated with estradiol in the presence of ionomycin, phorbol12-myristate 13-acetate, or okadaic acid under conditions where these agents do not synergize forERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient toinduce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute theresponse seen with growth factors. In this way, ERK-dependent and -independent signals may regulateMKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression (Cook, 1997).

In rat aortic smooth muscle cells (RASMC), pretreatment with forskolin inhibits the activation ofp42/44 isoforms of mitogen-activated protein kinase (MAP) kinase stimulated in response to lowconcentrations of PDGF. This correlates with a strong inhibition of PDGF-stimulated MEKand C-Raf-1 kinase activity. However, the effect of forskolin can be surmounted by increasing theconcentration of PDGF. Under such conditions forskolin is only effective against prolonged MAPkinase activation. The ability of forskolin to inhibit the late phase of MAP kinase activity is reversedby pretreatment of the cells with cycloheximide, suggesting the involvement of a protein synthesis step.This was not due to effects upstream of MAP kinase since PDGF-stimulated MEK activation isdecreased by cycloheximide, an effect potentiated by forskolin. Forskolin stimulates the induction ofthe dual specific phosphatase MAP kinase phosphatase-1 (MKP-1), although this effect is smallrelative to levels induced by PDGF and angiotensin II. However, PDGF stimulated induction of MKP-1is abolished by the protein kinase A inhibitor H89 and this correlates with the reversal offorskolin-mediated inhibition of PDGF-stimulated MAP kinase activity. These studies implicate a rolefor intracellular cyclic AMP in at least two aspects of MAP kinase signaling, including both theinhibition of Raf-1 activation and the induction of MKP-1 (Plevin, 1997).

Dual specificity phosphatases regulate Raf and MKK signaling

Inactivation of growth factor-regulated mitogen-activated protein (MAP) kinases (ERK1 and ERK2)has been proposed to occur in part through dephosphorylation by the dual specificity MAP kinasephosphatase-1 (MKP-1), an immediate early gene that is induced by mitogenic signaling. In this study,the effect of MKP-1 was examined on signaling components upstream of ERK1 and ERK2.Coexpression of MKK1 or MKK2 with MKP-1 results in 7-10-fold activation of mitogen-activatedprotein kinase kinase (MKK), which requires the presence of regulatory serine phosphorylation sites.Endogenous MKK1 and MKK2 are also activated upon MKP-1 expression. Raf-1, a direct regulatorof MKK1 and MKK2, is activated under these conditions, and a synergistic activation of MKK isobserved upon coexpression of Raf-1 and MKP-1. This effect does not appear to involve synthesis ofautocrine growth factors or the inhibition of basal extracellular signal-regulated kinase (ERK) activitybut is inhibited by a dominant negative Ras mutant, indicating that MKP-1 enhances Ras-dependentactivation of Raf-1 in a cell autonomous manner. This study demonstrates positive feedback regulationof Raf-1 and MKK by the MKP-1 immediate early gene and a potential mechanism for activatingRaf-1/MKK signaling pathways other than those involving ERK (Shapiro, 1998).

Mutation of dual specificity phosphatases

Externally regulated phosphatase (ERP or MKP-1) is a dual specificity phosphatase that has beenimplicated in the dephosphorylation of mitogen activated protein kinases (MAP kinases). MAP kinaseis activated in response to external signals and in turn phosphorylates proteins essential to the regulationof cell growth. To study the role of ERP/MKP-1 protein in mammalian development and its function insignal transduction, mice, embryonic stem (ES), cells and mouse embryo fibroblasts(MEFs) have been generated that are deficient in the ERP/MKP-1 protein. ERP/MKP-1-deficient mice are born at normalfrequency, are fertile and present no phenotypic or histologic abnormalities. MAP kinase activity andthe induction of c-fos mRNA is unaltered in MEFs lacking the ERP/MKP-1 protein, indicating noalteration of the MAP kinase pathway. In addition, ERP/MKP-1 deficient MEFs grow and enter DNAsynthesis at the same rate as control cells. These results demonstrate that the activity of ERP/MKP-1 isnot essential for embryo development and indicate that the lack of ERP/MKP-1 activity can becompensated by other phosphatases in vivo (Dorfman, 1996).

Dual specificity phosphatases, cell injury and apoptosis

In fibroblasts, serum stimulation has been shown to activate the immediate-early gene 3CH134 (MKP-1)encoding a dual specificity protein phosphatase that regulates mitogen-activated protein kinase. 3CH134 messenger RNA levels increase during recirculation following 30 minforebrain ischemia in the rat brain. In normal rat brains, 3CH134 messenger RNA is found mainly inneurons of the cortex and thalamus. At recirculation periods up to 1 h after 30 min ischemia, 3CH134messenger RNA increases in neurons and glial cells of all previously ischemic brain regions. After 3and 6 h recirculation, a prominent increase of 3CH134 messenger RNA is observed in the pyramidalcell layer of all sectors of the hippocampus and the granule cells of the dentate gyrus, whereas in theother brain regions messenger RNA levels return to control. Up to 6 h of recirculation, the spatialinduction pattern of 3CH134 is similar to the pattern observed for the immediate-early genes c-fosand c-jun. Within the hippocampus a similar pattern is observed for the heat shock protein hsp70messenger RNA. At 12 and 24 h after ischemia, increased levels of 3CH134 messenger RNApersist in hippocampal neurons; at the same time a delayed increase of 3CH134 messenger RNAis observed in large neurons of the thalamus and in glial cells in damaged regions of the striatum. Atlater survival periods, 3CH134 messenger RNA return to control levels. This study shows that themitogen-activated protein kinase phosphatase 3CH134 is induced in the brain after a period of globalischemia (Wiessner, 1995).

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activationof both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK)signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPKand p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38MAPK activity provides cytoprotection against UV-induced apoptosis, a U937 cell line conditionallyexpressing MKP-1 from the human metallothionein IIa promoter was established. Conditionalexpression of MKP-1 is found to abolish UV-induced SAPK and p38 MAPK activity, and inhibitsUV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 isalso found to inhibit other biochemical events associated with apoptosis, including activation ofcaspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase.These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within theapoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its abilityto inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect onUV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it wasfound that MKP-1 inhibits UV-induced SAPK activity, DNA fragmentation, and caspase-3 activationin a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibitUV-induced activation of SAPK, does not provide a similar cytoprotection against UV-inducedapoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection againstUV-induced apoptosis by inhibiting UV-induced SAPK activity (Franklin, 1998).

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