Activation Of Extracellular Regulated Protein Kinase Research Articles

ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3.

Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the “death” phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)–, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) overexpression results in a dramatic reduction of PM damaged neurons and nuclear condensation. In contrast, overexpression of constitutively active MEK potentiates PM damage and nuclear condensation. ERK-promoted cellular damage is independent of caspase-3. Persistent active ERK translocates to the nucleus, whereas caspase-3 remains in the cytoplasm. Antioxidants that reduced ERK activation and PM damage showed no effect on caspase-3 activation or DNA damage. These data identify ERK as an important executor of neuronal damage involving a caspase-3–independent mechanism.

Protein Kinase D Potentiates DNA Synthesis Induced by Gq-coupled Receptors by Increasing the Duration of ERK Signaling in Swiss 3T3 Cells

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.

Molecular mechanisms regulating protein kinase Czeta turnover and cellular transformation.

The regulation of protein kinase C (PKC)zeta in relation to its turnover, cell growth and transformation was investigated in Rat2 fibroblasts by over-expressing wild-type or mutant forms of PKCzeta. Deletion of the pseudosubstrate site (PSS) produced the most active mutant (PKCzeta Delta PSS), but mutants designed to mimic phosphorylated PKCzeta had lower specific activities in an in vitro assay. The mutant lacking phosphorylation at the Thr-560 site (T560A) had similar specific activity to wild-type PKCzeta. The T560A mutant also protected PKCzeta against proteolysis, whereas phosphorylation at Thr-410 targeted it towards proteosomal degradation. Blocking proteosomal degradation with lactacystin caused the accumulation of full-length PKCzeta Delta PSS, T410E, PKCzeta Delta PSS T410/560E, PKCzeta and T560A. Expressed PKCzeta activity was paralleled by extracellular-regulated protein kinase activation, increased cell division, serum-independent growth and focus formation. These foci were seen for cells expressing higher PKCzeta activity (PKCzeta Delta PSS, PKCzeta Delta PSS T410/560E and T560A mutants), but these fibroblasts did not show significant anchorage-independent growth. This work provides novel information concerning the role of the PSS and phosphorylation sites in regulating the activity and turnover of an atypical PKC and shows how this activity can induce cell transformation with respect to focus formation.

Pairing of forskolin and KCl increases differentiation of immortalized hippocampal neurons in a CREB Serine 133 phosphorylation-dependent and extracellular-regulated protein kinase-independent manner

Although cAMP response element binding protein (CREB)- and extracellular-regulated protein kinase (ERK)-mediated pathways have been linked to each other in neuronal differentiation, involvement of these in hippocampal neuronal cell line has not been defined. Using an immortalized hippocampal cell line, HiB5, we have tried a pairing of forskolin with KCl depolarization, which acts as an ERK and CREB kinase activator in hippocampal neurons, to investigate if an activation of ERK and phosphorylation of CREB at the critical regulatory site, serine 133 might be coupled in differentiation. Differentiation toward a neuronal phenotype was synergistically and markedly increased by the pairing of forskolin and KCl depolarization. The synergistic effect was accompanied by an increase in phosphorylation of CREB Ser-133, but not phosphorylation of ERK, and was not inhibited by MEK inhibitor, PD98059. These findings indicate that phosphorylation of the transcriptional factor CREB may function to facilitate differentiation of HiB5 cells.

SB203580 promotes EGF-stimulated early morphological differentiation in PC12 cell through activating ERK pathway.

MAP kinases have important role in PC12 cell differentiation, since the activities of both extracellular regulated protein kinase (ERK) and p38 have been indicated as necessary signal for PC12 cell differentiation. Epidermal growth factor (EGF) and NGF both activate ERK and p38 in PC12 cells, but only NGF trigger differentiation. It has been proposed that the duration of ERK activation determines the switch from proliferation to differentiation, since EGF causes more transient activation of ERK than NGF in PC12 cells. Here we report that treatment of PC12 cells with EGF in the presence of SB203580, a widely used p38 inhibitor, caused differentiation. The pro-differentiation effect of SB203580 in EGF-treated PC12 cells was found to be independent of its function of p38 inhibition but was through an effect on the ERK pathway that has been recently reported (Kalmes et al. [1999] FEBS Lett. 444: 71-74; Hall-Jackson et al. [1999] Onc. 18: 2047-2054). We found that SB203580 by itself did not affect the activity of ERK1/2 but significantly extended EGF-induced ERK activation in PC12 cells, which resulted in early morphological differentiation. Our data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated. Our data provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation.

Extracellular regulated kinase (ERK)-dependent regulation of sialomucin complex/rat Muc4 in mammary epithelial cells.

Sialomucin complex (SMC, rat Muc4) is a membrane mucin implicated in the protection of epithelia and the metastasis of some tumors. It is a heterodimeric complex, containing a mucin subunit with anti-adhesive activity and a transmembrane subunit with epidermal growth factor-like domains, one of which acts as an intramembrane ligand for ErbB2. Serum, insulin and insulin-like growth factor, but not epidermal growth factor, induce the expression of sialomucin complex in mammary epithelial cells. Induction correlates with sustained, but not transient, activation of extracellular-regulated protein kinase (ERK). MEK inhibitor U0126 blocked the induction, while activated MEK-1 transfected into a rat mammary adenocarcinoma cell line induced a sustained activation of ERK and up-regulated SMC/Muc4 expression. Northern and Western blotting indicated that up-regulation occurred concomitantly at the transcript and protein levels, both of which could be blocked by U0126. These results suggest that expression of SMC/Muc4 in mammary epithelial cells is regulated by selected growth factors through an ERK-dependent pathway at the transcript level.

Beta2-adrenergic receptor agonists and cAMP arrest human cultured airway smooth muscle cells in the G(1) phase of the cell cycle: role of proteasome degradation of cyclin D1.

Hyperplasia of airway smooth muscle (ASM) contributes to the airway hyperresponsiveness that characterizes asthma. We have investigated the relationship between cAMP-induced growth arrest of ASM cells and thrombin-stimulated, extracellular-regulated protein kinase (ERK) activity, cyclin D1, and the restriction protein retinoblastoma. The beta(2)-adrenergic receptor agonist albuterol (100 nM) inhibited DNA synthesis after incubation with ASM for periods as brief as 1 h when these coincided with the timing of the restriction point. Inhibition of thrombin-stimulated DNA synthesis by albuterol (1-100 nM), 8-bromo-cAMP (300 microM), or prostaglandin E(2) (1 microM) was accompanied by a reduction in cyclin D1 protein levels. The ERK kinase inhibitor PD98059 (3-30 microM) attenuated thrombin-stimulated ERK phosphorylation and activity and the increase in cyclin D1 protein levels, as did albuterol (1-100 nM) or 8-bromo-cAMP (300 microM). In contrast, neither albuterol (100 nM) nor PD98059 (30 microM) reduced cyclin D1 mRNA levels between 4 and 20 h after thrombin addition, which suggests that elevation of cAMP regulates cyclin D1 by a post transcriptional mechanism. The proteasome inhibitor MG132 (30 and 100 nM) and the calpain I inhibitor N-acetyl-Leu-Leu-leucinal (10 microM) attenuated the reduction in thrombin-stimulated cyclin D1 levels in ASM exposed to albuterol (100 nM), 8-bromo-cAMP (300 microM), or the phosphodiesterase inhibitor isobutylmethylxanthine (100 microM). Thus, the cAMP-induced arrest of ASM in the G(1) phase of the cell cycle is associated with a proteasomal degradation of cyclin D1 protein and a reduced protein retinoblastoma phosphorylation that prevents passage through the restriction point.

Effect of hydrogen peroxide on the metabolism of articular chondrocytes.

To examine the effect of hydrogen peroxide on chondrocyte metabolism. Bovine articular chondrocytes were used. Proteoglycan (PG) synthesis was measured with [35S] sulfate incorporation. For detection of apoptosis, the TdT-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V assay were used. Extracellular-regulated protein kinase (ERK) activity was measured using a mitogen-activated protein kinase assay system. Addition of hydrogen peroxide resulted in the inhibition of PG synthesis, apoptosis, and enhanced ERK activity. Hydrogen peroxide plays an important role in regulating the metabolism of chondrocytes.

Differential Activation of p38 Mitogen-activated Protein Kinase and Extracellular Signal-regulated Protein Kinases Confers Cadmium-induced HSP70 Expression in 9L Rat Brain Tumor Cells

We have reported that treatment with CdCl2 at 40-100 microM induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 microM) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38(MAPK)), while that in cells treated with a low concentration (60 microM) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 microM cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38(MAPK). By contrast, in 60 microM cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38(MAPK) and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38(MAPK) and ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.