One-way Cross-talk between p38MAPK and p42/44MAPK

157

109

We have identified a direct physical interaction between the stress signaling p38␣ MAP kinase and the mitogen-activated protein kinases ERK1 and ERK2 by affinity chromatography and coimmunoprecipitation studies. Phosphorylation and activation of p38␣ enhanced its interaction with ERK1/2, and this correlated with inhibition of ERK1/2 phosphotransferase activity. The loss of epidermal growth factor-induced activation and phosphorylation of ERK1/2 but not of their direct activator MEK1 in HeLa cells transfected with the p38␣ activator MKK6(E) indicated that activated p38␣ may sequester ERK1/2 and sterically block their phosphorylation by MEK1. Mitogen-activated protein (MAP)1 kinase modules are involved in the signal transduction of a wide variety of cellular responses in all eukaryotic organisms including proliferation, differentiation, and apoptosis (1). At least four distinct and parallel MAP kinase cascades have been identified, including extracellular signal-regulated kinases 1 and 2 (ERK1/2), the p38 MAP kinases, c-jun N-terminal or stress-activated protein kinases (JNK/SAPK), and ERK5/big MAP kinase 1 (BMK1). It is well established that ERK1/2 are typically stimulated by growth-related stimuli through the Raf1/B-MEK1/2-ERK1/2 protein kinase cascade. The JNK and p38 MAP kinases are primarily activated by stress-related signals such as heat and osmotic shock, UV irradiation, and proinflammatory cytokines by means of the MAP kinase kinases, MKK3,. Whereas the selective activation of distinct MAP kinase pathways in response to different extracellular stimuli has been extensively documented, there is increasing evidence for cross-talk between distinct MAP kinase pathways. A p38-dependent ERK1/2 activation was observed in several mammalian cell lines including the human embryonic kidney cell line HEK293 upon arsenite treatment (5). It was also found that inactivation of p38 by SB202190 treatment resulted in a delayed and prolonged activation of ERK1/2 in the human hepatoma cell line, HepG2 (6). In both cases, MEK1 was implicated in the activation of ERK1/2. Here we report that in HeLa and HEK293 cells, stress stimuli lead to an inhibition of ERK1/2 via p38␣. Phosphorylated p38␣ is capable of forming a complex with ERK1/2, and it prevents their phosphorylation by MEK1/2. EXPERIMENTAL PROCEDURESConstruction of pGEX-p38␣-The pGEX-p38␣ wild-type and p38␣ (AF) dominant negative mutant were constructed by subcloning the respective cDNA sequences into pGEX-4T vector (Amersham Pharmacia Biotech). The human p38 full-length sequences were obtained by digesting pcDNA3-flagp38␣ wild-type and p38␣ (AF) plasmids, a generous gift of Dr. J. Han (the Scripps Research Institute, La Jolla, CA). pGEX-ERK1 was obtained by subcloning human ERK1 cDNA fulllength sequence into pGEX-2T vector (Amersham Pharmacia Biotech). The resulting GST fusion protein constructs were verified by DNA sequencing (7).Expression of GST Fusion Proteins in Bacteria-GST fusion protein plasmids were transformed into DH5␣ bacteria. Expression of GST fusion prote...

Section: Resultssupporting

confidence: 82%

mentioning

confidence: 72%

Stress-induced Inhibition of ERK1 and ERK2 by Direct Interaction with p38 MAP Kinase

Zhang

Shi

Hampong

et al. 2001

157

109

“…However, when bombesin was present for more than 1 min this agonist caused MAP kinase activation. MAPK has been shown in a study on the human hepatoma cell line HepG2 (24). The rapid activation of p38 MAPK might also be involved in reorganization of actin cytoskeleton (21) and regulation of enzyme secretion.…”

Section: Discussionmentioning

confidence: 99%

Arachidonic Acid Modulates the Spatiotemporal Characteristics of Agonist-evoked Ca2+ Waves in Mouse Pancreatic Acinar Cells

Siegel

Sternfeld

González³

et al. 2001

MAPK and p42/44MAPK could also be observed in the presence of cholecystokinin (CCK), which also causes activation of cPLA 2 . ACh-and CCK-induced Ca 2؉ waves were slowed down when p38 MAPK (4,5). The speed of Ca 2ϩ waves depends on the type of agonist used for stimulation and to some extent also on the agonist concentration (6). The propagation rate can be modified by application of arachidonic acid (AA) or by activation of protein kinase C (PKC) with phorbol esters (6, 7). Both application of AA and activation of PKC leads to inhibition of CICR and thereby slows down spreading of Ca 2ϩ signals. Analysis of the propagation rate of secretagogueevoked Ca 2ϩ waves in pancreatic acinar cells can therefore be used to investigate coupling of hormone receptors to intracellular signal cascades that lead to endogenous production of AA and/or to activation of PKC. In previous studies we could demonstrate that in mouse pancreatic acinar cells bombesin receptors couple to phospholipase D (7), which produces diacylglycerol and thereby activates PKC, whereas high affinity CCK receptors couple to cytosolic phospholipase A 2 (cPLA 2 ) (6), an enzyme that leads to release of AA from membrane phospholipids. Bombesin-induced activation of PKC via phospholipase D-dependent diacylglycerol production, as well as CCK-induced formation of AA by cPLA 2 , takes place within the very first seconds after hormone application, before or during development of the initial inositol 1,4,5-trisphosphate-induced Ca 2ϩ signal in the luminal cell pole.In the present study we investigated the role of cPLA 2 in ACh-and bombesin-evoked Ca 2ϩ signaling. Our data indicate that in pancreatic acinar cells activation of cPLA 2 is an early event in ACh-but not in bombesin-evoked intracellular signaling. ACh-and CCK-induced activation of cPLA 2 involves MAP kinase activation. Furthermore, we demonstrate that secretagogue-induced Ca 2ϩ signaling in pancreatic acinar cells is modified by AA itself and not by metabolites of AA. MATERIALS AND METHODSCell Preparation-Adult male CD-1 mice (35-40 g) were sacrificed by cervical dislocation. The pancreas was removed and transferred into a "preparation buffer" consisting of (in mM): 130 NaCl, 4.7 KCl, 1.3 CaCl 2 , 1 MgCl 2 , 1.2 KH 2 PO 4 , 10 glucose, 0.2% (w/v) albumin, 0.01% (w/v) trypsin inhibitor, 10 HEPES, pH 7.4. Single acinar cells were isolated by enzymatic digestion with collagenase type V (30 units/ml, 10 min, 37°C; Sigma) as described previously (8). Enzymatic digestion was followed by mechanical dissociation of cells by gentle pipetting. The resulting cell suspension was centrifuged, and the cells were washed twice in preparation buffer without collagenase.Confocal Microscopy-Freshly prepared acinar cells were loaded with 4 M fluo-3/AM for 30 min at room temperature and then stored at 4°C. Experiments were performed within 4 h after cell isolation. For measurement of cytosolic Ca 2ϩ signals, cells were transferred to a perfusion chamber and were allowed to adhere to the glass coverslip for several minut...

“…For instance, in PANC-1, HL-60, L1210, and ECV304 cells, ERK1/2 activity or phosphorylation is increased by SB203580 or SB202190 (55)(56)(57)(58). In human skin fibroblasts, phorbol ester-and Raf-mediated induction of MEK1 and ERK1/2 are inhibited when p38 is activated by constitutively active MAPK kinase 3b, the kinase upstream of p38 (59), and in the human hepatoma cell line, HepG2, low density lipoprotein receptor mRNA and P-ERK1/2 are increased by SB treatment and reduced by constitutively active MAPK kinase 6b, another kinase upstream of p38 (60). These observations suggest a role for the p38 pathway in MEK-ERK-dependent processes.…”

Section: P38 Insulin and Erk Signalingmentioning

confidence: 99%

Insulin Signal Transduction Pathways and Insulin-induced Gene Expression

Keeton

Amsler

Venable

et al. 2002

Insulin regulates metabolic activity, gene transcription, and cell growth by modulating the activity of several intracellular signaling pathways. Insulin activation of one mitogen-activated protein kinase cascade, the MEK/ERK kinase cascade, is well described. However, the effect of insulin on the parallel p38 pathway is less well understood. The present work examines the effect of inhibiting the p38 signaling pathway by use of specific inhibitors, either alone or in combination with insulin, on the activation of ERK1/2 and on the regulation of gene transcription in rat hepatoma cells. Activation of ERK1/2 was induced by insulin and was dependent on the activation of MEK1, the kinase upstream of ERK in this pathway. Treatment of cells with p38 inhibitors also induced ERK1/2 activation/phosphorylation. The addition of p38 inhibitors followed by insulin addition resulted in a greater than additive activation of ERK1/2. The two genes studied, c-Fos and Pip92, are immediateearly genes that are dependent on the ERK1/2 pathway for insulin-regulated induction because the insulin effect was inhibited by pretreatment with a MEK1 inhibitor. The addition of p38 inhibitors induced transcription of both genes in a dose-dependent manner, and insulin stimulation of both genes was enhanced by prior treatment with p38 inhibitors. The ability of the p38 inhibitors to induce ERK1/2 and gene transcription, both alone and in combination with insulin, was abolished by prior inhibition of MEK1. These data suggest possible cross-talk between the p38 and ERK1/2 signaling pathways and a potential role of p38 in insulin signaling.