Decreased Mitogen-activated Protein Kinase Activation Research Articles

Phytoestrogen in cowpea (Vigna unguiculata L. Walp) (Fabaceae) extract reduces vaginal oxidative stress and increases proliferation of fibroblast in ovariectomized rats

Purpose: To evaluate the effects of a phytoestrogen extract of cowpea (Vigna unguiculata) on malondialdehyde (MDA) and superoxide dismutase (SOD) levels, mitogen-activated protein kinase (MAPK) activity, as well as proliferation of fibroblasts in ovariectomized rat vagina. This is with a view to identify the optimal dose of a phytoestrogen supplement for use during the menopausal period of women.
 Methods: Wistar rats (Rattus norvegicus) were divided into five groups: negative control, ovariectomy, and ovariectomy groups treated with 1.25, 2.5, or 5 mg/kg of cowpea extract, respectively. The three doses were used to identify the optimal dose required to reduce vaginal oxidative stress and increase fibroblast proliferation in ovariectomized rats. After treatment, the ovariectomized rat vagina was assessed for SOD and MDA levels, MAPK activity, and fibroblast proliferation.
 Results: Cowpea extract at a dose of 2.5 and 5 mg/kg caused significant reduction in SOD levels in the vaginal tissue of rats compared with control (p < 0.05). In contrast, cowpea extract at 1.25 mg/kg resulted in MDA levels similar to that of control rats (p < 0.05). Cowpea extract treatment had positive effects on SOD and MDA levels and on fibroblast proliferation. Increase in SOD levels in rat vagina was induced by increased MDA levels, which is characteristic of oxidative stress, while ovariectomy resulted in decreased MAPK activity.
 Conclusion: Overall, the cowpea extract has no significant effect on MAPK activity or fibroblast proliferation. However, it has a significant effect on SOD and MDA levels and on fibroblast proliferation at a dose of 2.5 mg/kg.

Sesamol suppresses the inflammatory response by inhibiting NF-κB/MAPK activation and upregulating AMP kinase signaling in RAW 264.7 macrophages.

Sesamol is a lignan isolated from sesame seed oil. In recent years, it was found that sesamol could decrease lung inflammation and lipopolysaccharide (LPS)-induced lung injury in rats. In this study, we investigated whether sesamol exhibited anti-inflammatory activity in LPS-stimulated macrophages. RAW 264.7 cells were treated with sesamol, then treated with LPS to induce inflammation. The levels of proinflammatory cytokines were analyzed with ELISA. The gene and protein expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and nuclear factor erythroid-2-related factor 2 (Nrf2) were evaluated with real-time PCR and Western blots, respectively. We also examined inflammatory signaling pathways, including nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Sesamol inhibited production of nitric oxide, prostaglandin E2 (PGE2), and proinflammatory cytokines. Sesamol markedly suppressed mRNA and protein expression of iNOS and COX-2. Sesamol enhanced the protective antioxidant pathway represented by Nrf2 and HO-1. Moreover, sesamol suppressed NF-κB transport into the nucleus and decreased MAPK activation, but it promoted adenosine monophosphate-activated protein kinase (AMPK) activation. These data suggested that sesamol ameliorated inflammatory and oxidative damage by upregulating AMPK activation and Nrf2 signaling and blocking the NF-κB and MAPK signaling pathways.

Anti-inflammatory effect of sophoraflavanone G isolated from Sophora flavescens in lipopolysaccharide-stimulated mouse macrophages

Sophoraflavanone G (SG; 5,7,D, 2',4'-tetrahydroxy-8-lavandulylflavanone) has been isolated from Sophora flavescens and found to be effective against bacteria and to decrease cyclooxygenase (COX)-2 expression in RAW 264.7 macrophage. However, the anti-inflammatory mechanisms of SG are not well understood. RAW 264.7 cells were pretreated with various concentrations of SG (2.5-20 μM) and inflammatory responses were induced with lipopolysaccharide. Using enzyme-linked immunosorbent assay, the levels of pro-inflammatory cytokines and prostaglandin E2 (PGE2) were determined. Western blot was used to examine the protein expression of inducible nitric oxide synthase (iNOS), COX-2, and heme oxygenase-1 (HO-1). To investigate the molecular mechanism, we analyzed inflammatory-associated signaling pathways, including nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK). SG inhibited the levels of nitric oxide and PGE2 and decreased the production of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor α. The expression of iNOS and COX-2 was also suppressed. However, SG increased HO-1 production in a concentration-dependent manner and significantly decreased MAPK activation and inhibited NF-κB subunit p65 proteins to translocate into the nucleus. These results suggest that SG has an anti-inflammatory effect, inhibiting pro-inflammatory cytokines and mediators production via interruption of the NF-κB and MAPK signaling pathways.

Abstract 4306: NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates MAPK activation and cellular protection against chemical stress.

Abstract NAD(P)H: quinone oxidoreductase (NQO1) is a cytosolic redox-regulating enzyme modulated by Nrf2-mediated stress signaling pathways, with an established role in regulation of stress-related transcription factors. In these studies we investigate the role of NQO1 in regulation of mitogen-activated protein kinase (MAPK) signaling pathways. Quantitative PCR and western blot studies showed that constitutive levels of several MAPK members are upregulated in NQO1-null mouse skin as well as in skin-derived keratinocytes, relative to WT. However, though total protein levels are increased, inducibility of these kinases by known MAPK inducers such as EGF and benzo[a]pyrene (BP) is significantly decreased in NQO1-null cells. NQO1 regulation of MAPK signaling was confirmed by studies showing that NQO1 knockdown by siRNA led to decreased MAPK activation, and overexpression of NQO1 protein led to increased MAPK activation by EGF. Similarly, downregulation of activity via dicoumarol caused loss of ERK phosphorylation, while NQO1 activation via t-BHQ caused increased ERK phosphorylation, which was blocked by MAPK pathway-specific inhibitors. To investigate the functional role of MAPK deactivation in skin-derived keratinocytes, studies with BP and menadione showed that lack of NQO1 expression led to increased susceptibility to ROS generation, and that this ROS generation causes increased toxicity. Taken together, these results show a significant role for NQO1 in regulation of MAPK signaling pathways, which ultimately lead to protection against ROS-mediated apoptosis. Citation Format: Brad A. Patrick, Anil K. Jaiswal. NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates MAPK activation and cellular protection against chemical stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4306. doi:10.1158/1538-7445.AM2013-4306

Abstract 318: NAD(P)H: quinone oxidoreductase 1 possesses a strong regulatory role in MAPK signaling pathways

Abstract NAD(P)H: quinone oxidoreductase (NQO1) is a cytosolic redox-regulating enzyme modulated by Nrf2-mediated stress signaling pathways, with an established role in regulation of stress-related transcription factors. In these studies we investigate the role of NQO1 in regulation of mitogen-activated protein kinase (MAPK) signaling pathways. Quantitative PCR and western blot studies showed that constitutive levels of several MAPK members are upregulated in NQO1-null mouse skin as well as in skin-derived keratinocytes, relative to wild type. However, though total protein levels are increased, inducibility of these kinases by known MAPK inducers such as EGF and benzo[a]pyrene (BP) is significantly decreased in NQO1-null cells. NQO1 regulation of MAPK signaling was confirmed by studies showing that NQO1 knockdown by siRNA led to decreased MAPK activation, and overexpression of NQO1 protein led to increased MAPK activation by EGF. Similarly, downregulation of activity via dicoumarol caused loss of ERK phosphorylation, while NQO1 activation via t-BHQ caused increased ERK phosphorylation, which was blocked by MAPK pathway-specific inhibitors. To investigate the functional role of MAPK deactivation in skin-derived keratinocytes, studies with BP and menadione showed that lack of NQO1 expression led to increased susceptibility to ROS generation, and that this ROS generation causes increased toxicity. Taken together, these results show a significant role for NQO1 in regulation of MAPK signaling pathways, which ultimately leads to protection against ROS-mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 318.

343 PHOSPHATIDYLINOSITOL 3-KINASE IS INVOLVED IN THE MAINTENANCE OF METAPHASE II ARREST IN PORCINE OOCYTES MATURED IN VITRO

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34cdc2 kinase activity. Next, the effect of 10 μM Ca2+ ionophore which was reported as inducing a transient decrease of p342+ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca2+ ionophore, both the MAPK and p34cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

Genetic evidence for the role of Erk activation in a lymphoproliferative disease of mice

Germline mutation of the linker for activation of T cells (LAT) gene at the phospholipase C-gamma1 (PLC-gamma1)-binding site leads to a fatal lymphoproliferative disease in mice. The hyperactivated T cells that develop in these mice have defective T-cell antigen receptor (TCR)-induced calcium flux but enhanced mitogen-activated protein kinase (MAPK) activation. We used genetic analysis to investigate genes whose products might suppress MAPK activation and lymphoproliferative disease in LAT mutant mice. B-lymphocyte adaptor molecule of 32 kDa (Bam32) is a known mediator of MAPK activation in B cells. We recently reported that in CD4(+) T cells, Bam32 deficiency decreased MAPK activation and specifically extracellular-signal-regulated kinase (Erk) signaling, following TCR stimulation. By crossing the Bam32 null mutation onto the LAT knock-in background, we found that the Bam32 null mutation delayed the onset and decreased the severity of lymphoproliferative disease in LAT knock-in mice. The pulmonary lymphocyte infiltration seen in LAT knock-in mice was also markedly decreased in double-mutant mice. Additionally, Erk activation was diminished in LAT knock-in Bam32 knockout CD4(+) T cells. To more accurately determine the role of Erk in this delay of lymphoproliferative disease, we also bred a transgenic, hypersensitive Erk allele (the Erk2 sevenmaker mutant) onto the LAT knock-in Bam32 knockout double-mutant background. These triple transgenic mice demonstrated a role for Erk activation in lymphoproliferative disease caused by the LAT knock-in mutation.

Diacylglycerol Kinase in T Cell Anergy

Defective Ras signaling contributes to T cell anergy, which occurs when T cells are presented with antigens in the absence of costimulatory signals and leads to T cells’ unresponsiveness to subsequent stimulation. T cell anergy is a key mechanism by which inappropriate immune responses are prevented. Previous work had shown that diacylglycerol (DAG)-mediated signaling was defective in anergic T cells and that abundance of the transcript encoding diacylglycerol kinase α (DGK-α) was decreased in activated T cells and increased in anergic T cells. Two groups (Zha et al . and Olenchock et al .) extend these observations and show that DGK-α, an enzyme that converts DAG to phosphatic acid, contributes to T cell anergy. Zha et al . showed that introduction of constitutively active Ras, one of the components downstream of DAG, restored T cell responsiveness [interleukin-2 (IL-2) production and mitogen-activated protein kinase (MAPK) phosphorylation] to cells previously rendered anergic. Expression of the Dgka gene was increased in anergic T cells in the system used by Zha et al ., and overexpression of DGK-α in resting T cells diminished recruitment of the Ras guanine nucleotide exchange factor RasGRP1 to the plasma membrane, decreased IL-2 production, and decreased MAPK activation in response to stimulation. Olenchock et al . used cells from Dgka −/− mice to show that T cells from these animals failed to undergo anergy in response to T cell receptor activation in the absence of costimulatory signals. Together, these two reports demonstrate a key role in DAG metabolism in promoting T cell anergy (see Mueller). What remains to be determined is how DGK activity is regulated to prevent excessive activity in cells that receive an activating signal and to promote activity in cells that receive a signal that produces anergy. Y. Zha, R. Marks, A. W. Ho, A. C. Peterson, S. Janardhan, I. Brown, K. Praveen, S. Stang, J. C. Stone, T. F. Gajewski, T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-α. Nat. Immunol. 7 , 1166-1173 (2006). [PubMed] B. A. Olenchock, R. Guo, J. H. Carpenter, M. Jordan, M. K. Topham, G. A. Koretzky, X.-P. Zhong, Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat. Immunol. 7 , 1174-1181 (2006). [PubMed] D. L. Mueller, Linking diacylglycerol kinase to T cell anergy. Nat. Immunol. 7 , 1132-1134 (2006). [PubMed]

Mitogen-activated protein kinase kinase inhibitor suppresses cyclin B1 synthesis and reactivation of p34cdc2 kinase, which improves pronuclear formation rate in matured porcine oocytes activated by Ca2+ ionophore.

To investigate the role of mitogen-activated protein (MAP) kinase kinase (MEK)/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes, MEK activity, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were assayed in mature porcine oocytes after treatment with different concentrations of Ca2+ ionophore. A high concentration of Ca2+ ionophore (50 microM) rapidly reduced MEK activity in oocytes for up to 8 h of culture. MEK activity in the 10-microM treatment group was significantly higher. The low concentration treatment transiently decreased p34cdc2 kinase activity but did not affect MAP kinase activity and ultimately induced reactivation of p34cdc2 kinase via the synthesis of cyclin B1. On the other hand, treatments of a high concentration of Ca2+ ionophore or a low concentration of Ca2+ ionophore plus MEK inhibitor, U0126, linearly decreased MAP kinase activity following the decrease of p34cdc2 kinase activity; most of these oocytes formed pronuclei. These results suggest that decreasing MAP kinase activity is essential to maintaining low p34cdc2 kinase activity resulting from the degradation of cyclin B via a Ca(2+)-dependent pathway; lower activities of both MAP kinase and p34cdc2 kinase induce normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.

Differential regulation of the mitogen-activated protein kinases by pathogenic and nonpathogenic mycobacteria.

Mycobacteria are the etiologic agents of numerous diseases which account for significant morbidity and mortality in humans and other animal species. Many mycobacteria are intramacrophage pathogens and therefore the macrophage response to infection, which includes synthesis of cytokines such as tumor necrosis factor alpha (TNF-alpha) and production of nitric oxide, has important consequences for host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection or how pathogenic mycobacteria may modulate the macrophage responses. Using primary murine bone marrow macrophages, we established that p38 and extracellular signal-regulated kinases 1 and 2 of the mitogen-activated protein kinase (MAPK) pathways are activated upon infection with different species of mycobacteria. However, we observed decreased MAPK activity over time in macrophages infected with pathogenic Mycobacterium avium strains relative to infections with nonpathogenic mycobacteria. Furthermore, macrophages infected with M. avium produced lower levels of TNF-alpha, interleukin 1beta, and inducible nitric oxide synthase 2 than macrophages infected with nonpathogenic species. Inhibitor studies indicate that the MAPKs are required for the Mycobacterium-mediated induction of these effector proteins. Our data indicate that MAPKs are activated in macrophages upon invasion by mycobacteria and that this activation is diminished in macrophages infected with pathogenic strains of M. avium, resulting in decreased production of important immune effector proteins. The decreased MAPK activation associated with M. avium infections suggests a novel point of immune intervention by this mycobacterial species.

Depletion of Raf-1 protooncogene by geldanamycin causes apoptosis in human luteinized granulosa cells

Objective: To investigate the hypothesis that epidermal growth factor (EGF) signaling in luteinized granulosa cells works through Raf-1 and mitogen-activated protein (MAP) kinases and that depletion of Raf-1 by geldanamycin will inhibit the signaling pathway and cause apoptosis. Design: Laboratory study. Setting: University of Minnesota. Patient(s): Human luteinized granulosa cells from IVF patients. Intervention(s): The cells were treated with vehicle (DMSO), 0.5 μM of geldanamycin, 10 ng/mL of EGF, and geldanamycin + EGF. Main Outcome Measure(s): Radiochemical MAP kinase assay, Western blotting, confocal microscopy, and flow cytometry. Result(s): Geldanamycin treatment depleted Raf-1 and lowered MAP kinase activity in luteinized granulosa cells. EGF treatment increased MAP kinase phosphorylation and translocation of the phosphorylated MAP kinase to the nucleus. Geldanamycin blocked this effect. Cleavage of caspase-3, the executioner protein in apoptosis, into an active 17 kD fragment was observed by Western blotting in geldanamycin-treated cells. Finally, by flow cytometry we observed significantly increased percentages of subdiploid apoptotic nuclei in geldanamycin-treated cells. Conclusion(s): In human luteinized granulosa cells, EGF works through Raf-1, and MAP kinase and depletion of Raf-1 by geldanamycin resulted in decreased MAP kinase activity, increased activated caspase-3, and, ultimately, apoptosis.

Binding and signaling of angiotensin-(1–7) in bovine kidney epithelial cells involves the AT 4 receptor

Angiotensin-(1–7) decreased mitogen-activated protein (MAP) kinase (Erks) activation in cultured Mardin–Darby bovine kidney (MDBK) epithelial cells. Also, saturable, high-affinity 125I-angiotensin-(1–7) binding was detected in MDBK cell membranes. Together, the data suggested the possible presence of an angiotensin-(1–7) receptor. However, ligand structure-binding studies revealed that angiotensin-(3–7) and AT 4 receptor ligands competed with high-affinity for 125I-angiotensin-(1–7) binding. Furthermore, angiotensin-(3–7) and AT 4 receptor ligands decreased MAP kinase activation in MDBK cells. These results demonstrate that NH 2-terminal-deleted metabolites of angiotensin-(1–7) can bind with high affinity to the AT 4 receptor and regulate the MAP kinase/Erk signaling pathway in renal epithelial cells.

Differential Effects of cAMP in Neurons and Astrocytes: ROLE OF B-RAF

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes, with important implications for both survival and proliferation.

Endotoxin activation of mitogen-activated protein kinase in THP-1 cells; diminished activation following endotoxin desensitization.

The signal transduction events occurring in monocytes in response to endotoxin (LPS) stimulation are incompletely delineated, although pertussis toxin (PT)-sensitive G proteins and the mitogen-activated protein kinase (MAPK) cascade have been implicated. Cellular desensitization in response to 18-h pre-exposure to 1 microgram/mL LPS alters signal transduction pathways of cellular activation and decreases production of certain inflammatory mediators such as thromboxane (Tx)B2, the stable metabolite of TxA2. We hypothesized that LPS stimulation of the human monocyte cell line THP-1 occurs via MAPK activation, and that LPS desensitization, induced by pre-exposure to LPS, is associated with altered signaling through the MAPK cascade. Involvement of a specific MAPK, ERK, in LPS-stimulated TxB2 production was further tested using a specific MAPK cascade inhibitor, PD98059 (PD). PD inhibited LPS and phorbol myristate acetate (PMA)-stimulated ERK activation as demonstrated by immunoblots using anti-activated ERK antibodies. PD significantly inhibited LPS and PMA-stimulated TxB2 synthesis to non-detectable levels, suggesting an involvement of MAPK in LPS-stimulated activation. Because PT-sensitive G proteins mediate LPS-stimulated signal transduction, their role in MAPK activation was tested. Pretreatment with PT inhibited basal and LPS-stimulated, but not PMA-stimulated ERK activation. Activation of ERK after LPS desensitization was also assessed. LPS pre-exposure resulted in a profound decrease in LPS-stimulated activation of ERK, but did not affect PMA activation of ERK. These data implicate the involvement of the MAPK cascade in LPS-stimulated activation of THP-1 cells and suggest coupling of Gi proteins and MAPKs in LPS-stimulated events. LPS desensitization is associated with decreased MAPK activation, but does not impair MAPK activation by PMA. Thus, LPS desensitization appears to selectively alter signal transduction upstream of ERK.

Control of PHAS-I by Insulin in 3T3-L1 Adipocytes: SYNTHESIS, DEGRADATION, AND PHOSPHORYLATION BY A RAPAMYCIN-SENSITIVE AND MITOGEN- ACTIVATED PROTEIN KINASE-INDEPENDENT PATHWAY

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-I eukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H6]PHAS-I when the [H6]PHAS-I.eIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-I.eIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70S6K. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.