Schizosaccharomyces pombe Spk1 is a tyrosine-phosphorylated protein functionally related to Xenopus mitogen-activated protein kinase.

Mitogen-activated protein kinase (MAPK) and its direct activator, MAPK kinase (MAPKK), have been suggested to play a pivotal role in a variety of signal transduction pathways in higher eukaryotes. The fission yeast Schizosaccharomyces pombe carries a gene, named spk1, whose product is structurally related to vertebrate MAPK. Here we show that Spk1 is functionally related to Xenopus MAPK. (i) Xenopus MAPK partially complemented a defect in the spk1- mutant. An spk1- diploid strain could not sporulate, but one carrying Xenopus MAPK could. (ii) Both Spk1 and Xenopus MAPK interfered with sporulation if overexpressed in S. pombe cells. (iii) Spk1 underwent tyrosine phosphorylation as does Xenopus MAPK. Tyrosine phosphorylation of Spk1 appeared to be dependent upon mating signals because it occurred in homothallic cells but not in heterothallic cells. Furthermore, this phosphorylation was diminished in a byr1 disruptant strain, suggesting that spk1 lies downstream of byr1, which encodes a MAPKK homolog in S. pombe. Taken together, the MAPKK-MAPK cascade may be evolutionarily conserved in signaling pathways in yeasts and vertebrates.     

Anthrax lethal factor causes proteolytic inactivation of mitogen-activated protein kinase kinase

A search of the National Cancer Institute's Anti-Neoplastic Drug Screen for compounds with an inhibitory profile similar to that of the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059 yielded anthrax lethal toxin. Anthrax lethal factor was found to inhibit progesterone-induced meiotic maturation of frog oocytes by preventing the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Similarly, lethal toxin prevented the activation of MAPK in serum stimulated, ras-transformed NIH3T3 cells. In vitro analyses using recombinant proteins indicated that lethal factor proteolytically modified the NH2-terminus of both MAPKK1 and 2, rendering them inactive and hence incapable of activating MAPK. The consequences of this inactivation upon meiosis and transformed cells are also discussed.     

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.     

Control of the cell morphology and the S phase entry by mitogen-activated protein kinase kinase. A regulatory role of its n-terminal region

The mitogen-activated protein kinase kinase (MAPKK)/MAP kinase (MAPK) cascade plays an important role in the growth control of mammalian cells. We have found that expression of constitutively active MAPKK induces rapid morphological changes of fibroblastic cells, which are accompanied by disruption of stress fibers and disappearance of focal adhesions. These changes took place under the conditions that inhibited cellular Ras function, suggesting a linkage between the MAPK cascade and the control of cell morphology. We further show that constitutively active MAPKK can induce expression of endogenous Fos protein, an immediately early gene product, and cause the S phase entry of G0-arrested cells. Finally, expression of the N-terminal fragment of MAPKK which encompasses the nuclear export signal sequence and the MAPK-binding site blocked both the serum-induced S phase entry of quiescent cells and the oncogenic Ras-induced morphological changes. All these results demonstrate that MAPKK is one of key molecules involved in the control of both cell morphology and cell proliferation and suggest an important role for the N-terminal region of MAPKK in the regulation of the MAPK signaling.     

Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases that are widely distributed in eukaryotic cells. Studies in the last decade revealed that MAPK cascade plays pivotal roles in regulating the meiotic cell cycle progression of oocytes. In mammalian species, activation of MAPK in cumulus cells is necessary for gonadotropin-induced meiotic resumption of oocytes, while MAPK activation is not required for spontaneous meiotic resumption. After germinal vesicle breakdown (GVBD), MAPK is involved in the regulation of microtubule organization and meiotic spindle assembly. The activation of this kinase is essential for the maintenance of metaphase II arrest, while its inactivation is a prerequisite for pronuclear formation after fertilization or parthenogenetic activation. MAPK cascade interacts extensively with other protein kinases such as maturation-promoting factor, protein kinase A, protein kinase C, and calmodulin-dependent protein kinase II, as well as with protein phosphatases in oocyte meiotic cell cycle regulation. The cross talk between MAPK cascade and other protein kinases is discussed. The review also addresses unsolved problems and discusses future directions.     

Graded mitogen-activated protein kinase activity precedes switch-like c-Fos induction in mammalian cells

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-or-nothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.     

Requirement for mitogen-activated protein kinase in cerebellar long term depression

The mitogen-activated protein kinase (MAPK) cascade has been shown to play an essential role in regulation of cell proliferation and cell differentiation. Although mammalian MAPKs are most abundantly expressed in postmitotic and terminally differentiated neuronal cells, their function in the central nervous system is still largely undefined. We present evidence here for a role of the MAPK cascade in cerebellar long term depression (LTD), which is a widely studied form of synaptic plasticity in mammalian brain. In cultured Purkinje cells, LTD is known to be induced by iontophoretic application of glutamate and depolarization of Purkinje cells. We found that MAPK was activated in Purkinje cells by treatment of primary cultures of rat embryonic cerebella with glutamate and a depolarization-inducing agent, KCl. Application of PD98059, a specific inhibitor of MAPK kinase (MAPKK/MEK), inhibited both the activation of MAPK and the induction of LTD in Purkinje cells. Furthermore, the induction of LTD was completely blocked by introduction into Purkinje cells of anti-active MAPK antibody, which was found to specifically and potently inhibit the activity of MAPK. These results suggest that postsynaptic activation of the MAPK cascade is essential for the induction of cerebellar LTD.     

Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.

MAP kinase kinase (MAPKK) has been identified as a protein factor that can induce phosphorylation and activation of inactive MAP kinase in vitro. In this study, we produced an anti-Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro. Microinjection of this antibody into immature oocytes prevented progesterone-induced MAP kinase activation. Moreover, progesterone-induced histone H1 kinase activation and germinal vesicle breakdown (GVBD) were inhibited in the oocytes injected previously with this antibody. Furthermore, when a bacterially expressed Mos was introduced into immature oocytes, Mos-induced MAP kinase activation and GVBD were blocked in the oocytes injected with the anti-MAPKK antibody. These results show that MAPKK is responsible for the activation of MAP kinase in vivo and that the MAPKK/MAP kinase cascade plays a pivotal role in the MPF activation during the oocyte maturation process.     

Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings

The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.     

Signal convergence through the lenses of MAP kinases: paradigms of stress and hormone signaling in plants

Common mechanisms plants use to translate the external stimuli into cellular responses are the activation of mitogen-activated protein kinase (MAPK) cascade. These MAPK cascades are highly conserved in eukaryotes and consist of three subsequently acting protein kinases, MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK) which are linked in various ways with upstream receptors and downstream targets. Plant MAPK cascades regulate numerous processes, including various environmental stresses, hormones, cell division and developmental processes. The number of MAPKKs in Arabidopsis and rice is almost half the number of MAPKs pointing important role of MAPKKs in integrating signals from several MAPKKKs and transducing signals to various MAPKs. The cross talks between different signal transduction pathways are concentrated at the level of MAPKK in the MAPK cascade. Here we discussed the insights into MAPKK mediated response to environmental stresses and in plant growth and development.     

Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.     

Isolation of two members of the rat MAP kinase kinase gene family

Mitogen-activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c-Raf-1. It is, however, unclear whether the signals originating from c-Raf-1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of 5 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK. may form a large gene family.     

Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase.

The mitogen-activated protein kinase (MAPK) cascade consisting of MAPK and its direct activator, MAPK kinase (MAPKK), is essential for signaling of various extracellular stimuli to the nucleus. Upon stimulation, MAPK is translocated to the nucleus, whereas MAPKK stays in the cytoplasm. It has been shown recently that the cytoplasmic localization of MAPKK is determined by its nuclear export signal (NES) in the near N-terminal region (residues 33-44). However, the mechanism determining the subcellular distribution of MAPK has been poorly understood. Here, we show that introduction of v-Ras, active STE11 or constitutively active MAPKK can induce nuclear translocation of MAPK in mammalian cultured cells. Furthermore, we show evidence suggesting that MAPK is localized to the cytoplasm through its specific association with MAPKK and that nuclear accumulation of MAPK is accompanied by dissociation of a complex between MAPK and MAPKK following activation of the MAPK pathway. We have identified the MAPK-binding site of MAPKK as its N-terminal residues 1-32. Moreover, a peptide encompassing the MAPK-binding site and the NES sequence of MAPKK has been shown to be sufficient to retain MAPK to the cytoplasm. These findings reveal the molecular basis regulating subcellular distribution of MAPK, and identify a novel function of MAPKK as a cytoplasmic anchoring protein for MAPK.     

Signaling through MAP kinase networks in plants

Protein phosphorylation is the most important mechanism for controlling many fundamental cellular processes in all living organisms including plants. A specific class of serine/threonine protein kinases, the mitogen-activated protein kinases (MAP kinases) play a central role in the transduction of various extra- and intracellular signals and are conserved throughout eukaryotes. These generally function via a cascade of networks, where MAP kinase (MAPK) is phosphorylated and activated by MAPK kinase (MAPKK), which itself is activated by MAPKK kinase (MAPKKK). Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as response to various stresses. In plants, MAP kinases are represented by multigene families and are organized into a complex network for efficient transmission of specific stimuli. Putative plant MAP kinase cascades have been postulated based on experimental analysis of in vitro interactions between specific MAP kinase components. These cascades have been tested in planta following expression of epitope-tagged kinases in protoplasts. It is known that signaling for cell division and stress responses in plants are mediated through MAP kinases and even auxin, ABA and possibly ethylene and cytokinin also utilize a MAP kinase pathway. Most of the biotic (pathogens and pathogen-derived elicitors) including wounding and abiotic stresses (salinity, cold, drought, and oxidative) can induce defense responses in plants through MAP kinase pathways. In this article we have covered the historical background, biochemical assay, activation/inactivation, and targets of MAP kinases with emphasis on plant MAP kinases and the responses regulated by them. The cross-talk between plant MAP kinases is also discussed to bring out the complexity within this three-component module.     

Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis.

Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1). Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation. In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases. The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways. These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes. We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs. One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X. laevis. XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle. Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2. Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator. The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2. The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase. Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates.     

Mitogen-activated protein kinase signaling in plants under abiotic stress

Mitogen-activated protein kinase cascade is evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. This cascade consists essentially of three components, a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK connected to each other by the event of phosphorylation. These kinases play various roles in intra- and extra-cellular signaling in plants by transferring the information from sensors to responses. Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as responses to various stresses. MAPK signaling has also been associated with hormonal responses. In plants, MAP kinases are represented by multigene families and are involved in efficient transmission of specific stimuli and also involved in the regulation of the antioxidant defense system in response to stress signaling. In the current review we summarize and investigate the participation of MAPKs as possible mediators of various abiotic stresses in plants.Key words: abiotic stress, cross talk, mitogen-activated protein kinases, heat map, MAPK signaling, signal transduction, stress signaling     

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.     

Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.     

Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.     

Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase.

p38 MAPK is a Ser/Thr protein kinase activated by various inflammatory cytokines and a variety of stress stimuli. It is involved in many physiological processes, including the production of inflammatory cytokines. We have previously reported the design and synthesis of a series of pyridinylimidazole compounds that are selective inhibitors of p38 MAPK. These compounds, exemplified by SB 203580, are exceptionally effective in cell-based assays, including the inhibition of inflammatory cytokine production. SB 203580 is widely used as a tool to dissect the role of p38 MAPK in various physiological processes. It has previously been established that SB 203580 acts primarily to block the catalytic activity of p38 MAPK. However, it has been suggested that in cells, the compounds could also inhibit p38 MAPK activation by virtue of their ability to bind to the inactive enzyme. We undertook careful studies to definitively demonstrate that treatment with SB 203580 had no effect on Thr(180) and Tyr(182) phosphorylation, and hence activation of p38 in vivo. SB 203580, however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK. This was observed regardless of stimuli or cell type. Identical results were obtained when the p38 MAPK cascade was partially reconstituted in vitro. Thus, our data clearly indicate that SB 203580 specifically inhibits the activity of p38 MAPK but not its activation by upstream MAPKK.