Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase signal transduction pathway

Neurotrophic factor deprivation causes apoptosis by a mechanism that requires macromolecular synthesis. This fact suggests that gene expression is necessary to achieve cell death. To identify mRNA that is expressed in apoptotic cells we used subtractive hybridization with cDNA prepared from neuronal pheochromocytoma cells. Monoamine oxidase (MAO) expression was increased in cells during nerve growth factor withdrawal-induced apoptosis. The increased apoptosis and induction of MAO was prevented by inhibition of the p38 mitogen-activated protein (MAP) kinase pathway. MAO may contribute to the apoptotic process because inhibition of MAO activity suppressed cell death. Together, these data indicate that MAO may be a target of pro-apoptotic signal transduction by the p38 MAP kinase pathway.

Mitotic Phosphorylation of Golgi Reassembly Stacking Protein 55 by Mitogen-activated Protein Kinase ERK2

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.

Selective response of ternary complex factor Sap1a to different mitogen-activated protein kinase subgroups.

Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear. Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions.

Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Ultrasensitivity in the mitogen-activated protein kinase cascade.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved series of three protein kinases implicated in diverse biological processes. Here we demonstrate that the cascade arrangement has unexpected consequences for the dynamics of MAPK signaling. We solved the rate equations for the cascade numerically and found that MAPK is predicted to behave like a highly cooperative enzyme, even though it was not assumed that any of the enzymes in the cascade were regulated cooperatively. Measurements of MAPK activation in Xenopus oocyte extracts confirmed this prediction. The stimulus/response curve of the MAPK was found to be as steep as that of a cooperative enzyme with a Hill coefficient of 4-5, well in excess of that of the classical allosteric protein hemoglobin. The shape of the MAPK stimulus/ response curve may make the cascade particularly appropriate for mediating processes like mitogenesis, cell fate induction, and oocyte maturation, where a cell switches from one discrete state to another.

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.

Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.

RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.