Transcriptional activation of dual specificity phosphatases: a feedback loop

Phorbol ester tumor promoters, such as phorbol 12-myristate 13-acetate (PMA), are potent activators of extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase (SAPK), and p38 mitogen-activated protein kinase (MAPK) in U937 human leukemic cells. These kinases are regulated by the reversible dual phosphorylation of conserved threonine and tyrosine residues. The dual specificity protein phosphatase MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate and inactivate ERK2, SAPK, and p38 MAPK in transient transfection studies. PMA treatment induces MKP-1 protein expression in U937 cells, which is detectable within 30 min with maximal levels attained after 4 h. This time course coincides with the rapid inactivation of PMA-induced SAPK activity, but not ERK2 phosphorylation, which remains elevated for up to 6 h. To examine directly the role of MKP-1 in the regulation of these protein kinases in vivo, a U937 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, by titrating the levels of MKP-1 expression from the human metallothionein IIa promoter it was found that p38 MAPK and SAPK are much more sensitive to inhibition by MKP-1 than ERK2. This differential substrate specificity of MKP-1 can be functionally extended to nuclear transcriptional events because PMA-induced c-Jun transcriptional activity is more sensitive to inhibition by MKP-1 than either Elk-1 or c-Myc. Conditional expression of MKP-1 also abolishes the induction of endogenous MKP-1 protein expression in response to PMA treatment. This negative feedback regulatory mechanism is likely due to MKP-1-mediated inhibition of ERK2, since studies utilizing the MEK1/2 inhibitor PD98059 suggest that ERK2 activation is required for PMA-induced MKP-1 expression. These findings suggest that ERK2-mediated induction of MKP-1 may play an important role in preferentially attenuating signaling through the p38 MAPK and SAPK signal transduction pathways (Franklin, 1997).

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and MKP-2 are two members of a recently described family of dual specificity phosphatases that are capable of dephosphorylating p42/p44MAPK. Overexpression of MKP-1 or MKP-2 inhibits MAP kinase-dependent intracellular signaling events and fibroblast proliferation. MKP-1 and MKP-2 are not expressed in quiescent cells, 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 protein detectable up to 14 h after serum addition. Induction of MKP-1 and (to a lesser extent) MKP-2 temporally correlates with p42/p44MAPK inactivation. To analyze the contribution of the MAP kinase cascade to MKP-1 and MKP-2 induction, CCL39 cells were examined that had been transformed with either v-ras or a constitutively active direct upstream activator of MAP kinase, mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1[SD/SD] mutant). In both cell models, MKP-1 and MKP-2 are constitutively expressed, with MKP-2 being prevalent. CCL39 cells expressing an estradiol-inducible deltaRaf-1::ER chimera, activation of Raf alone is sufficient to induce MKP-1 and MKP-2. The role of the MAP kinase cascade in MKP induction was highlighted by the MKK-1 inhibitor PD 098059, which blunts both the activation of p42/p44MAPK and the induction of MKP-1 and 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 protein kinases A and C. Thus, activation of the p42/p44MAPK cascade promotes the induction of MKP-1 and MKP-2, which may then attenuate p42/p44MAPK-dependent events in an inhibitory feedback loop (Brondello, 1997).

Insulin signaling involves the transient activation/inactivation of various proteins by a cycle of phosphorylation/dephosphorylation. This dynamic process is regulated by the action of protein kinases and protein phosphatases. One family of protein kinases that is important in insulin signaling is the mitogen-activated protein (MAP) kinases, whose action is reversed by specific MAP kinase phosphatases (MKPs). Insulin stimulation of Hirc B cells overexpressing the human insulin receptor results in increased MKP-1 mRNA levels. MKP-1 mRNA increases in a dose-dependent manner to a maximum of 3- to 4-fold over basal levels within 30 min, followed by a gradual return to basal. The mRNA induction does not require the continuous presence of insulin. The induction of MKP-1 protein synthesis follows MKP-1 mRNA induction; MKP-1 protein is maximally expressed after 120 min of insulin stimulation. MKP-1 mRNA induction by insulin requires insulin receptor tyrosine kinase activity, since overexpression of an altered insulin receptor with impaired intrinsic tyrosine kinase activity prevents mRNA induction. Forskolin, (Bu)2-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP increase the MKP-1 mRNA content moderately above basal. These agents also augment the insulin-stimulated expression of MKP-1 mRNA. However, in some cases the increase in MKP-1 mRNA expression is less than additive. Nevertheless, these results indicate that multiple signaling motifs might regulate MKP-1 expression and suggest another mechanism for the attenuation of insulin-stimulated MAP kinase activity by cAMP. Overexpression of MKP-1 in Hirc B cells inhibits both insulin-stimulated MAP kinase activity and MAP kinase-dependent gene transcription. The results of these studies lead to the conclusion that insulin regulates MKP-1 and strongly suggest 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 in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate has little effect on the early peak of ERK1 activity but potentiates the sustained phase. Cycloheximide also potentiates ERK1 activation in Rat-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-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, it was speculated that the 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 selective inhibitor of Gi-protein-coupled signaling pathways, reduces LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER has little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC plays little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression is blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, a strong synergy of MKP-1 expression is observed when cells are stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents do not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-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 of p42/44 isoforms of mitogen-activated protein kinase (MAP) kinase stimulated in response to low concentrations of PDGF. This correlates with a strong inhibition of PDGF-stimulated MEK and C-Raf-1 kinase activity. However, the effect of forskolin can be surmounted by increasing the concentration of PDGF. Under such conditions forskolin is only effective against prolonged MAP kinase activation. The ability of forskolin to inhibit the late phase of MAP kinase activity is reversed by 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 is decreased by cycloheximide, an effect potentiated by forskolin. Forskolin stimulates the induction of the dual specific phosphatase MAP kinase phosphatase-1 (MKP-1), although this effect is small relative to levels induced by PDGF and angiotensin II. However, PDGF stimulated induction of MKP-1 is abolished by the protein kinase A inhibitor H89 and this correlates with the reversal of forskolin-mediated inhibition of PDGF-stimulated MAP kinase activity. These studies implicate a role for intracellular cyclic AMP in at least two aspects of MAP kinase signaling, including both the inhibition 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 kinase phosphatase-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-activated protein 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 regulator of MKK1 and MKK2, is activated under these conditions, and a synergistic activation of MKK is observed upon coexpression of Raf-1 and MKP-1. This effect does not appear to involve synthesis of autocrine growth factors or the inhibition of basal extracellular signal-regulated kinase (ERK) activity but is inhibited by a dominant negative Ras mutant, indicating that MKP-1 enhances Ras-dependent activation of Raf-1 in a cell autonomous manner. This study demonstrates positive feedback regulation of Raf-1 and MKK by the MKP-1 immediate early gene and a potential mechanism for activating Raf-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 been implicated in the dephosphorylation of mitogen activated protein kinases (MAP kinases). MAP kinase is activated in response to external signals and in turn phosphorylates proteins essential to the regulation of cell growth. To study the role of ERP/MKP-1 protein in mammalian development and its function in signal 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 normal frequency, are fertile and present no phenotypic or histologic abnormalities. MAP kinase activity and the induction of c-fos mRNA is unaltered in MEFs lacking the ERP/MKP-1 protein, indicating no alteration of the MAP kinase pathway. In addition, ERP/MKP-1 deficient MEFs grow and enter DNA synthesis at the same rate as control cells. These results demonstrate that the activity of ERP/MKP-1 is not essential for embryo development and indicate that the lack of ERP/MKP-1 activity can be compensated 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 min forebrain ischemia in the rat brain. In normal rat brains, 3CH134 messenger RNA is found mainly in neurons of the cortex and thalamus. At recirculation periods up to 1 h after 30 min ischemia, 3CH134 messenger RNA increases in neurons and glial cells of all previously ischemic brain regions. After 3 and 6 h recirculation, a prominent increase of 3CH134 messenger RNA is observed in the pyramidal cell layer of all sectors of the hippocampus and the granule cells of the dentate gyrus, whereas in the other brain regions messenger RNA levels return to control. Up to 6 h of recirculation, the spatial induction pattern of 3CH134 is similar to the pattern observed for the immediate-early genes c-fos and c-jun. Within the hippocampus a similar pattern is observed for the heat shock protein hsp70 messenger RNA. At 12 and 24 h after ischemia, increased levels of 3CH134 messenger RNA persist in hippocampal neurons; at the same time a delayed increase of 3CH134 messenger RNA is observed in large neurons of the thalamus and in glial cells in damaged regions of the striatum. At later survival periods, 3CH134 messenger RNA return to control levels. This study shows that the mitogen-activated protein kinase phosphatase 3CH134 is induced in the brain after a period of global ischemia (Wiessner, 1995).

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of 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 SAPK and p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provides cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 is found to abolish UV-induced SAPK and p38 MAPK activity, and inhibits UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 is also found to inhibit other biochemical events associated with apoptosis, including activation of caspase-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 the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect on UV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it was found that MKP-1 inhibits UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit UV-induced activation of SAPK, does not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity (Franklin, 1998).