Sustained MAP Kinase Activation Research Articles

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.

Subcellular receptor redistribution and enhanced microspike formation by a Ret receptor preferentially recruiting Dok

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret(Dok+)) with little or no remaining interactions with Shc and Frs2. Ret(Dok+) displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret(WT). We show that early events after ligand stimulation of Ret(Dok+) include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret(Dok+) receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.

The EphA8 receptor induces sustained MAP kinase activation to promote neurite outgrowth in neuronal cells

Recent studies in our laboratory demonstrate that ligand-mediated activation of the EphA8 receptor critically regulates cell adhesion and migration. In this report, we show that the EphA8 receptor induces neurite outgrowth in NG108-15 cells in the absence of ligand stimulation. Using various deletion mutants lacking specific intracytoplasmic regions, we confirm that the tyrosine kinase domain of EphA8 is important for inducing neurite outgrowth. However, the tyrosine kinase activity of EphA8 is not crucial for neurite outgrowth induction. Treatment with various inhibitors further reveals that the mitogen-activated protein kinase (MAPK) signaling pathway is critical for neurite outgrowth induced by EphA8. Consistent with these results, EphA8 expression induced a sustained increase in the activity of MAPK, whereas ligand-mediated EphA8 activation had no further modulatory effects on MAP kinase activity. Additionally, activated MAPK relocalized from the cytoplasm to the nucleus in response to EphA8 transfection. These results collectively suggest that the EphA8 receptor is capable of inducing a sustained increase in MAPK activity, thereby promoting neurite outgrowth in neuronal cells.

P0620 TUMOR NECROSIS FACTOR (TNF) INDUCES MATRIX METALLOPROTEINASE-9 (MMP-9) EXPRESSION AND ACTIVITY IN MOUSE COLON

Introduction: TNF is a cytokine that regulates growth, migration, differentiation and apoptosis in normal and diseased intestinal epithelium through complex signaling mechanisms. MMP-9 belongs to a family of endopeptidases involved in tissue injury, repair, and tumorigenesis. MMP-9 is elevated in patients with inflammatory bowel disease (IBD), and its inhibition in murine models of colitis ameliorates disease severity. Since sustained MAP kinase activation is necessary for epithelial cell growth inhibition by TNF and MMP-9 expression, we investigated TNF regulation of MMP-9 expression and the involved TNF receptors in intestinal epithelium. Methods: Young adult mouse colon (YAMC), TNFR1−/ −, or TNFR2−/ − mouse colon epithelial (MCE) cells were treated with TNF (100 ng/ml) or EGF (10 ng/ml) for various times (1–24 h). Cellular lysates, total RNA and conditioned media were analyzed for MMP-9 expression by Western blot analysis, 2-D Difference Gel Electrophoresis and MALDI-TOF, or gene microarray and gelatin zymography, respectively. MMP-9 was studied by immunodetection in mouse colon after TNF injection (104 U/mouse) or in 2 models of IBD with increased endogenous TNF production, IL 10−/ − colitis and mice receiving water with dextran sulfate sodium (5% wt/vol) for 7 days. All were compared to appropriate controls. Results: TNF increased MMP-9 expression 6-fold in YAMC cells at 4 hours by gene microarray and proteomic screening assays. Furthermore, conditioned media derived from TNF-treated cells showed a 20-fold increase in both the active and pro-forms of MMP-9 and a decrease in MMP-2 expression by gelatin zymography. By comparison, EGF enhanced MMP-2 by 3–5-fold but had no effect on MMP-9 expression. TNF increased MMP-9 expression in TNFR2−/ −MCE but not TNFR1−/ −MCE cells, indicating TNFR1 is required for this effect. Treatment of mice with exogenous TNF increased MMP-9 expression in the epithelial and surrounding stromal cells at the base of colonic glands. Induced colitis, either in IL 10−/ − deficiency or exogenous dextran sulfate sodium administration, increased MMP-9 expression similarly. MMP-9 was increased in IL 10−/ − mice with colitis more than 20-fold compared to background control mice. Conclusion: In the mouse colon, MMP-9 expression is increased by TNF or colitis through TNFR1 in epithelial and surrounding stromal cells. Increased expression of MMP-9 through TNFR1 may contribute to epithelial damage, defective epithelial remodeling and tumorigenesis in IBD.

Multifaceted roles of TNF‐α in myoblast destruction: A multitude of signal transduction pathways

In catabolic conditions, such as cancer cachexia, a balance favouring a cytokine environment culminates in muscle destruction. Utilising an in vitro model to mimic muscle wasting, we elucidate here the multifaceted roles that one such cytokine, TNF-alpha, invokes in the degeneration process. Treatment of C2 skeletal myoblasts with TNF-alpha not only suppresses morphological and biochemical differentiation, but following an initial wave of proliferation, and of survival (24 h), induces apoptosis. Investigating the mechanisms underlying these diverse actions of TNF-alpha, we demonstrate that cell replication is dependent on rapid and sustained activation of MAP kinase. Map kinase is not, however, central to the death process, which is associated with a progressive rise in caspase-8 activity, and is accompanied by sustained activation of JNK1 and transient activation of JNK2. Caspase inhibition caused a dose responsive reduction in cell death, while inhibition of the JNKs caused a significant increase in apoptosis. We further report that PI3 kinase is not involved in conferring early protection against TNF-alpha-induced death. By contrast, inhibition of NF-kappaB in the presence of TNF-alpha culminates in increased cell cycle progression, decreased gadd45beta expression and significant and precociously increased cell death, when compared with TNF-alpha alone. Our results begin to characterise the mechanisms underlying the acute mitogenic and anti-apoptotic roles of TNF-alpha, which appear to be defined by a balance between MAP kinase, Jun kinase (JNK), NF-kappaB and gadd45beta. They establish that inhibition of any one of these molecules, as may occur following caspase activation, could eliminate vital stem cells required for skeletal muscle regeneration during chronic catabolic conditions.

Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel

Accumulation of phosphorylated isoforms of the microtubule-associated protein tau is one hallmark of affected neurons in Alzheimer’s disease (AD). This increase has been attributed to increased kinase or decreased phosphatase activity. Prior studies indicate that one of the kinases that phosphorylates tau (mitogen-activated protein kinase, or MAP kinase) does so at least in part indirectly within intact neuronal cells by phosphorylating and activating the L-voltage-sensitive calcium channel. Resultant calcium influx then fosters tau phosphorylation via one or more calcium-activated kinases. We demonstrate herein that treatment of differentiated SH-SY-5Y human neuroblastoma with the phosphatase inhibitor okadaic acid (OA) similarly may increase tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel. OA increased phospho-tau as indicated by increased immunoreactivity towards an antibody (PHF-1) directed against paired helical filaments from AD brain. This increase was blocked by co-treatment with the channel antagonist nimodipine. OA treatment increased channel phosphorylation. The increases in calcium influx, PHF-1 immunoreactivity and channel phosphorylation were all attenuated by co-treatment with PD98059, which inhibits MAP kinase activity, suggesting that OA mediates these effects at least in part via sustained activation of MAP kinase. These findings underscore that divergent and convergent kinase and phosphatase activities regulate tau phosphorylation.

A Role for Focal Adhesion Kinase in Hyluronan-Dependent MMP-2 Secretion in a Human Small-Cell Lung Carcinoma Cell Line, QG90

Hyluronan (HA), a nonsulfated high-molecular mass glycoaminoglycan, has been assigned as a clinical marker for the progression of various tumors. We found that HA stimulation of QG90, a cell line derived from human small-cell lung carcinoma, activates the secretion of matrix metalloproteinase-2 (MMP-2) in a focal adhesion kinase (FAK)-dependent manner. HA stimulation of QG90 cells activated MMP-2 secretion in a time-dependent manner. Larger sizes of HA seemed to have higher activities than smaller size one in MMP-2 secretion. Under HA stimulation, tyrosine phosphorylation of cellular proteins including FAK was activated. By use of antisense oligonucleotide to FAK, we found that FAK signaling was required for the activation of MMP-2 secretion and for the sustained activation of MAP kinase by HA treatment. These results strongly suggest that FAK-MAPK signaling is involved, at least in part, in HA-dependent activation of MMP-2 secretion in QG90 cells.

Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells

MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. We report here that p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restored MyoD function and enhanced MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines could activate the p38 MAPK in RMS cells, however, these stimuli did not promote differentiation, possibly because they activated p38 MAPK only transiently and they also activated JNK, which could antagonize differentiation. Thus, the selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors.

Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells.

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21CiP1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 and cell cycle re-entry. The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent MAPK signalling pathway but that expression of late AP-1 genes and cyclin D1 requires that this pathway be persistently activated. The results suggest that the duration of receptor signalling and therefore MAPK activation is a key determinant of qualitative changes in gene expression during cell cycle re-entry.

Differential regulation of MAP kinase, p70(S6K), and Akt by contraction and insulin in rat skeletal muscle.

To study the effects of contractile activity on mitogen-activated protein kinase (MAP kinase), p70 S6 kinase (p70(S6K)), and Akt kinase signaling in rat skeletal muscle, hindlimb muscles were contracted by electrical stimulation of the sciatic nerve for periods of 15 s to 60 min. Contraction resulted in a rapid and transient activation of Raf-1 and MAP kinase kinase 1, a rapid and more sustained activation of MAP kinase and the 90-kDa ribosomal S6 kinase 2, and a dramatic increase in c-fos mRNA expression. Contraction also resulted in an apparent increase in the association of Raf-1 with p21Ras, although stimulation of MAP kinase signaling occurred independent of Shc, IRS1, and IRS2 tyrosine phosphorylation or the formation of Shc/Grb2 or IRS1/Grb2 complexes. Insulin was considerably less effective than contraction in stimulating the MAP kinase pathway. However, insulin, but not contraction, increased p70(S6K) and Akt activities in the muscle. These results demonstrate that contraction-induced activation of the MAP kinase pathway is independent of proximal steps in insulin and/or growth factor-mediated signaling, and that contraction and insulin have discordant effects with respect to the activation of the MAP kinase pathway vs. p70(S6K) and Akt. Of the numerous stimulators of MAP kinase in skeletal muscle, contractile activity emerges as a potent and physiologically relevant activator of MAP kinase signaling, and thus activation of this pathway is likely to be an important molecular mechanism by which skeletal muscle cells transduce mechanical and/or biochemical signals into downstream biological responses.

An anchorage-dependent signal distinct from p42/44 MAP kinase activation is required for cell cycle progression

Most normal cells require both mitogens and integrin-mediated attachment for growth. It is generally accepted that the p42/p44 MAP kinase module, which can be activated by both growth factors and adhesion, plays a critical role in G0 to S phase progression of quiescent cells. Studies on various cultured fibroblasts have shown that removal of anchorage leads to cell cycle arrest in G1 and it has been proposed that adhesion-dependent G1 progression requires the joint regulation of p42/p44 MAP kinase by integrins and growth factors. In quiescent CCL39 lung fibroblasts, MAP kinase activation in response to serum becomes compromised when cells are placed in suspension. Under these conditions, serum-stimulated cells arrest their growth in mid-G1 with reduced cyclin D1 expression and increased p21Cip/Waf1 expression, as compared to their attached counterparts. To determine whether a casual link exists between suboptimal activation of MAP kinase in non-adherent cells and the observed G1 block, we used a variant of CCL39 stably expressing an estrogen-inducible activated-Raf-1 construct (deltaRaf-1:ER). We found that even strong and sustained activation of MAP kinase with estradiol, in addition to serum, is not able to boost cyclin D1 expression levels or stimulate hyperphosphorylation of pRb in suspended CCL39-deltaRaf-1:ER cells. These results indicate that p42/p44 MAP kinase activation is not a limiting factor for G1 to S phase transit in absence of anchorage. Thus, at least one adhesion-mediated signalling event, distinct from MAP kinase activation is required for maximal cyclin D1 induction and hyperphosphorylation of pRb.

Binding of Shp2 tyrosine phosphatase to FRS2 is essential for fibroblast growth factor-induced PC12 cell differentiation.

FRS2 is a lipid-anchored docking protein that plays an important role in linking fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein (MAP) kinase signaling pathway. In this report, we demonstrate that FRS2 forms a complex with the N-terminal SH2 domain of the protein tyrosine phosphatase Shp2 in response to FGF stimulation. FGF stimulation induces tyrosine phosphorylation of Shp2, leading to the formation of a complex containing Grb2 and Sos1 molecules. In addition, a mutant FRS2 deficient in both Grb2 and Shp2 binding induces a weak and transient MAP kinase response and fails to induce PC12 cell differentiation in response to FGF stimulation. Furthermore, FGF is unable to induce differentiation of PC12 cells expressing an FRS2 point mutant deficient in Shp2 binding. Finally, we demonstrate that the catalytic activity of Shp2 is essential for sustained activation of MAP kinase and for potentiation of FGF-induced PC12 cell differentiation. These experiments demonstrate that FRS2 recruits Grb2 molecules both directly and indirectly via complex formation with Shp2 and that Shp2 plays an important role in FGF-induced PC12 cell differentiation.

Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation.

Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level within 2 h. Conversely, the induction by 10(-12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a low EGF concentration, suggesting that both EGF concentrations activated distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.

Sustained MAP kinase activation in vessel organ culture model of hypertension

s; 16th Meeting of the French Hypertension Society; Palais des Congrès, Paris, 12–13 December 1996

V-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 - 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 - 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.

Truncated, desensitization-defective neurokinin receptors mediate sustained MAP kinase activation, cell growth and transformation by a Ras-independent mechanism.

We have used the neurokinin NK-2 receptor as a model to examine how receptor desensitization affects cellular responses. The liganded receptor transiently activates phospholipase C (PLC) and is rapidly phosphorylated on Ser/Thr residues in its C-terminal domain. Mutant receptors lacking this domain mediate persistent activation of PLC. We now show that, in transfected Rat-1 cells, mutant receptor mediates ligand-induced DNA synthesis, morphological transformation and growth in soft agar, whereas wild-type (wt) receptor does not. Wt receptor causes only transient MAP kinase activation. In contrast, MAP kinase activation by mutant receptor is sustained for >4 h. Neither wt nor mutant receptor couples to Ras activation. Downregulation of protein kinase C (PKC) has little effect on MAP kinase activation, DNA synthesis and transformation. Mutant receptors also promote stronger protein tyrosine phosphorylation and stress fibre formation than does wt receptor. Thus, C-terminal truncation allows the NK-2 receptor to signal sustained MAP kinase activation, cell growth and transformation by a Ras- and PKC-independent mechanism. Our results reveal the importance of the C-terminal 'desensitization domain' in suppressing the oncogenic potential of a prototypic PLC-coupled receptor.

Activation of MAP kinase by the LHRH receptor through a dual mechanism involving protein kinase C and a pertussis toxin-sensitive G protein

The LHRH receptor in αT3-1 gonadotrope cells was shown to bring about a marked and sustained activation of MAP kinase. This response was prevented by protein kinase C inhibition or down-regulation and could be partially mimicked by phorbol ester. Additional evidence for inhibition of this response by pertussis toxin and partial mimicry by mastoparan (in a pertussis toxin-sensitive manner) provides the first evidence for G i G o -mediated signal transduction by the LHRH receptor. This dual mechanism of MAP kinase activation appears to be exceptional amongst the G protein-linked receptors that have been investigated.

Differentiation of PC12 cells in response to a cAMP analogue is accompanied by sustained activation of mitogen-activated protein kinase: Comparison with the effects of insulin, growth factors and phorbol esters

It has been proposed previously that the sustained activation of mitogen-activated protein kinase may be necessary for the differentiation of PC12 cells. Differentiation of PC12 cells is induced by many extracellular agonists including nerve growth factor (NGF) and cyclicAMP analogues, but not epidermal growth factor (EGF), insulin or phorbol esters. Our results demonstrate that: (i) 8-(4-chlorophenylthio)-cyclicAMP (CPT-cAMP) activates MAP kinase; this raises the possibility that the MAP kinase pathway may be activated by agents that act through adenylate cyclase; (ii) NGF and CPT-cAMP as well as phorbol esters promote sustained activation of MAP kinase. This suggests that while sustained MAP kinase activation may be associated with differentiation it may not be sufficient, and that other as yet unidentified parallel pathways may be involved.