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Gene clusters contributing to processes such as cell growth and pathogenicity are often controlled by two-component signal transduction systems (TCSs). Specific inhibitors against TCS systems work differently from conventional antibiotics, and developing them into new drugs that are effective against various drug-resistant bacteria may be possible. Furthermore, inhibitors of TCSs that control virulence factors may reduce virulence without killing the pathogenic bacteria. Previous TCS inhibitors targeting the kinase domain of the histidine kinase sensor suffered from poor selectivity. Recent TCS inhibitors, however, target the sensory domains of the sensors blocking the quorum sensing system, or target the essential response regulator. These new targets are introduced, together with several specific TCSs that have the potential to serve as effective drug targets., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase (MAPK) kinase (MAPKK, also known as MEK), a direct activator for MAPK/extracellular signal-regulated kinase, localizes to the cytoplasm excluded from the nucleus during signal transmission. This nuclear exclusion of MAPKK is directed by its nuclear export signal (NES), but its physiological significance has been unknown. We have found that disruption of the NES dramatically potentiates the ability of constitutively active MAPKK to induce morphological changes and malignant transformation of fibroblastic cells. Readdition of the NES sequence reversed the effects induced by the NES disruption. Moreover, we observed that a dramatic increase of activated MAPK in the nucleus was induced by the NES-disrupted MAPKK and that coexpression of MAPK phosphatase-1 (CL-100) or a kinase negative form of MAPK counteracted the phenotypes induced by the NES-disrupted MAPKK, indicating the crucial role of MAPK in the responses. These findings reveal a novel regulatory role of the NES of MAPKK that may be essential for proper signal transductions.

Stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, is thought to play a key role in a variety of cellular responses. To date, SEK1/MKK4, one of the MAP kinase kinase (MAPKK) family of molecules, is the only SAPK/JNK kinase that has been cloned. Here we have cloned, identified and characterized a novel member of the mammalian MAPKKs, designated MKK7. MKK7 is most similar to the mediator of morphogenesis, hemipterous (hep), in Drosophila. Immunochemical studies have identified MKK7 as one of the major SAPK/JNK-activating kinases in osmotically shocked cells. While SEK1/MKK4 can activate both the SAPK/JNK and p38 subgroups of the MAPK superfamily, MKK7 is specific for the SAPK/JNK subgroup. MKK7 is activated strongly by tumour necrosis factor alpha (TNFalpha) as well as by environmental stresses, whereas SEK1/MKK4 is not activated by TNFalpha. Column fractionation studies have shown that MKK7 is a major activator for SAPK/JNK in the TNFalpha-stimulated pathway. Moreover, we have found that overexpression of MKK7 enhances transcription from an AP-1-dependent reporter construct. Thus, MKK7 is an evolutionarily conserved MAPKK isoform which is specific for SAPK/JNK, is involved in AP-1-dependent transcription and may be a crucial mediator of TNFalpha signalling.

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.

We have previously shown that osmotic stress activates both the mitogen-activated protein kinase (MAPK) cascade and the stress-activated protein kinase (SAPK, also known as JNK) cascade in rat fibroblastic 3Y1 cells and rat PC12 cells. Here, we show that treatment of these cells with sodium arsenite, a chemical compound that mimics the effects of heat shock, or anisomycin, a protein synthesis inhibitor, induces activation of SAPKs potently. These chemical compounds also stimulated the activity of SEK1/MKK4/JNKK, SAPK activator, and the activity of MEKK, SEK1 activator. Expression of a dominant negative mutant of Ras blocked the anisomycin-induced activation of SAPK and SEK1, but did not affect markedly the arsenite-induced or heat shock-induced activation in PC12 cells. The osmotic-stress-induced activation of SAPK was insensitive to the expression of a dominant negative Ras, but was partly sensitive to down-regulation of protein kinase C. These results suggest the existence of Ras-dependent and Ras-independent activation pathways for the SAPK cascade triggered by environmental stresses including chemical stress in PC12 cells. Cell staining with a specific anti-SAPK serum showed that SAPKs were present in both the cytoplasm and the nucleus under normal conditions, and became located mainly in the nucleus after osmotic stress or ultraviolet treatment, suggesting the nuclear translocation of SAPKs.

MAP kinase kinase (MAPKK) is a dual specificity protein kinase that phosphorylates and activates MAP kinase in vivo. In this study, four mouse monoclonal single-chain Fv (scFv) antibodies (Y1-6, Y1-7, Y3-6, and Y3-11) that can specifically bind to Xenopus MAPKK were isolated from combinatorial scFv-displaying phage libraries. Three scFv clones (Y1-6, Y1-7, and Y3-6) were shown to efficiently inhibit MAPKK activity in vitro. Point mutation (D98K) at VH-CDR3 of one (Y1-6) of these three clones markedly reduced its neutralizing activity. The wild-type scFv (Y1-6) inhibited the Mos-induced MAP kinase activation and germinal vesicle breakdown when injected into immature Xenopus oocytes, whereas the mutant scFv, Y1-6 (D98K), did not. The three neutralizing scFv clones (Y1-6, Y1-7, and Y3-6) were shown to bind to NH2-terminal residues 1-23 of Xenopus MAPKK, whereas the epitope of a Y3-11 clone with no neutralizing activity was shown to lie between residues 33 and 67 of MAPKK. Furthermore, a synthetic peptide (the N16 peptide) corresponding to residues 2-17 of MAPKK suppressed the neutralizing activity of the wild-type Y1-6, and a rabbit polyclonal antibody against the N16 peptide was found to possess a strong neutralizing activity against MAPKK. These results demonstrate that the neutralizing antibodies characterized here inhibit the kinase activity of MAPKK by binding to the NH2-terminal segment of MAPKK.

Mitogen-activated protein kinase (MAPK) is activated in cytoplasm in response to extracellular signals and then is translocated to nucleus. A directed activator for MAPK, MAPK kinase (MAPKK), stays in cytoplasm to transmit the signal from the plasma membrane to MAPK. Here we show that MAPKK contains a short amino acid sequence in the N-terminal region (residues 32-44), which acts as a nuclear export signal (NES) and thus is required for cytoplasmic localization of MAPKK. This NES sequence of MAPKK, like that of protein kinase inhibitor of cAMP-dependent protein kinase or Rev, is rich in leucine residues, which are crucial for the NES activity. Furthermore, the NES peptide of protein kinase inhibitor, as well as the NES peptide of MAPKK, inhibited the nuclear export of ovalbumin conjugated to the NES peptide of MAPKK. These results may suggest a common mechanism of nuclear export using a general leucine-rich NES.

Mitogen-activated protein kinase kinase (MAPKK) is a dual-specificity protein kinase which phosphorylates and activates mitogen-activated protein kinase (MAPK). cDNAs encoding two isoforms of MAPKK, MAPKK1 and MAPKK2 (also known as MEK1 and MEK2), have been cloned in mammalian cells. To analyze the characteristics of MAPKK1 and MAPKK2 individually, we have produced specific anti-MAPKK serum against each isoform. MAPKK1 and MAPKK2 have apparent molecular masses of 45 kDa and 47 kDa, respectively, on SDS/polyacrylamide gel electrophoresis. In mouse tissues, MAPKK1 was highly enriched in brain, while MAPKK2 was present relatively evenly. In rat fibroblastic 3Y1 cells, epidermal growth factor (EGF) treatment induced activation of both MAPKK1 and MAPKK2. Immunoprecipitation experiments have shown that the time courses of activation and deactivation of both isoforms of MAPKK were superimposed. In PC12 cells, both MAPKK1 and MAPKK2 were activated in response to nerve growth factor (NGF) as well as EGF, and the time courses of activation and deactivation of both isoforms were indistinguishable from each other in the NGF-stimulated cells and also in the EGF-stimulated cells. Furthermore, localization of both MAPKK1 and MAPKK2 in the cytoplasm was unchanged in response to EGF and NGF. Thus, the same or quite similar mechanisms may operate in the regulation of the activation and deactivation of two isoforms of MAPKK, and both kinases might have redundant functions when expressed in the same cell.

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPK alpha in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK.

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.

The product of the c-mos proto-oncogene functions not only as an initiator of oocyte maturation but also as a component of cytostatic factor that causes the natural arrest of the unfertilized egg at the second meiotic metaphase. It has been shown that Mos can phosphorylate and activate mitogen-activated protein (MAP) kinase kinase (MAPKK) in vitro, leading to activation of MAP kinase. In this study, by using an anti-MAPKK antibody that can specifically inhibit Xenopus MAPKK activity, we have shown that MAPKK mediates the cytostatic factor activity of Mos. Coinjection of this anti-MAPKK antibody with the bacterially expressed Mos protein into a two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-induced MAP kinase activation. The analysis of individual embryos indicated that the degree of the cleavage arrest was correlated with the extent of the MAP kinase activation in the Mos- and the Mos/antibody-injected embryos. These observations suggest the involvement of a signal transmission pathway consisting of Mos, MAPKK, and MAP kinase in the metaphase arrest.

Mitogen-activated protein (MAP) kinase and its direct activator, MAP kinase kinase (MAPKK), comprise the MAPKK/MAP kinase cascade, which may play a pivotal role in a variety of intracellular signal transduction pathways from yeast to human. Vertebrate MAPKK, a dual-specificity kinase, is activated by serine phosphorylation catalyzed by upstream serine/threonine kinases, MAPKK kinases (MAPKK-Ks). MAPKK is, on the other hand, threonine phosphorylated by MAP kinase, although a physiological role of this MAP kinase-mediated phosphorylation of MAPKK is unknown. Biochemical fractionation of extracts from Xenopus mature oocytes revealed two major and one minor peaks for the MAPKK-K activity. One of the major peaks contained a proto-oncogene product c-Mos, while the other peaks did not. These observations, together with a recent finding that several MAPKK-Ks such as Raf-1 and MEKK may function within a cell, suggest a diversity of MAPKK-Ks. A variety of extracellular signals converge at the MAPKK/MAP kinase cascade through different MAPKK-Ks and elicit a wide spectrum of cellular responses. Therefore, mechanisms that control activation of the MAP kinase cascade temporally and spatially may be important for specification of cellular responses.

Xenopus mitogen-activated protein kinase kinase (MAPKK) previously inactivated with protein phosphatase 2A can be reactivated by serine phosphorylation catalyzed by a partially purified MAPKK kinase (MAPKK-K), and is phosphorylated by MAPK on a threonine residue. The sequence analysis of a threonine-phosphorylated tryptic peptide of Xenopus MAPKK from mature oocytes suggested that Thr388 is phosphorylated in vivo. A mutant MAPKK that has Thr388 changed to Ala (T388A-MAPKK) was not phosphorylated by purified MAPK, indicating that Thr388 is phosphorylated by MAPK. We then produced and analysed MAPKKs mutated at potential serine phosphorylation sites (S218A-MAPKK and S222A-MAPKK). The wild-type MAPKK (WT-MAPKKK), T388A-MAPKK and a kinase-deficient (K97S)-MAPKK were phosphorylated efficiently by MAPKK-Ks purified from Xenopus eggs, and WT-MAPKK and T388A-MAPKK became activated. In contrast, neither S218A-MAPKK nor S222A-MAPKK was phosphorylated and activated efficiently by the Xenopus MAPKK-Ks. Similarly, WT-MAPKK, but not S218A-MAPKK or S222A-MAPKK, was activated efficiently by an active Raf-1 immunoprecipitate. However, when the recombinant STE11, a putative MAPKK-K in S. cerevisiae, was used as a source of MAPKK-K, S218A-MAPKK as well as WT-MAPKK, but not S222A-MAPKK, was phosphorylated and activated. Furthermore, replacement of Ser222 with an acidic residue (S222E) elevated substantially the basal kinase activity of MAPKK, while replacement of Ser218 (S218E) did not. These results may suggest an essential role for Ser222 phosphorylation in activating Xenopus MAPKK.

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.

The platelet-activating factor (PAF) was seen to potently activate mitogen-activated protein (MAP) kinase and MAP kinase kinase through the cloned guinea pig PAF receptor stably expressed in Chinese hamster ovary (CHO) cells. Both 42- and 44-kDa MAP kinases were activated and tyrosine-phosphorylated in response to PAF. The PAF receptor also triggered the production of inositol phosphates and the release of arachidonic acid and inhibited cyclic AMP accumulation. Differential inhibitory effects of pertussis toxin (PTX) on these signals suggested that the PAF receptor couples to both PTX-sensitive and -insensitive G proteins in CHO cells. MAP kinase and MAP kinase activations were partially regulated by PTX-sensitive G proteins. The PAF receptor did not trigger any detectable increase in the GTP form of Ras under the conditions in which the human insulin receptor expressed in the same parent CHO cells potently increased the level. Since these agonists induced comparable MAP kinase activations through cognate receptors, Ras seems to play different roles in MAP kinase activation by the two different classes of receptors. The activation of MAP kinase by the cloned PAF receptor may explain part of the mechanisms underlying PAF-induced differentiation and proliferation in non-inflammatory cells.

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.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.

Mitogen-activated protein (MAP) kinases are activated by combined tyrosine and threonine phosphorylation catalysed by MAP kinase kinase, a novel class of protein kinases with dual specificity for both tyrosine and serine/threonine. MAP kinase kinase is turned on by serine/threonine phosphorylation catalysed by an immediate upstream kinase. The MAP kinase cascade appears to be conserved during evolution and thus might play an essential role in diverse intracellular signaling processes from yeasts to vertebrates.

Xenopus 45-kDa mitogen-activated protein (MAP) kinase kinase (MAPKK) is a serine/threonine/tyrosine kinase, which activates MAP kinase (MAPK) by phosphorylating its threonine and tyrosine residues. MAPKK is active only when its threonine and/or serine residues are phosphorylated. We have identified from Xenopus eggs two protein kinases responsible for phosphorylation of MAPKK. The two kinases are separated by Sephacryl S-300 gel filtration chromatography. The higher molecular weight kinase phosphorylates MAPKK previously dephosphorylated and inactivated by phosphatase 2A treatment on mainly serine and slightly threonine residues, and reactivates the MAPKK, and is thus assumed to work as MAPKK kinase (MAPKKK) in vivo. The lower molecular weight kinase, identified as MAPK, phosphorylates the dephosphorylated MAPKK on mainly threonine and faintly serine residues, but does not reactivate the MAPKK activity. As Xenopus MAPKK contains a single phosphorylation consensus sequence (PXT388P) for MAPK in the C-terminal region, this T388 residue may be a major phosphorylation site catalyzed by MAPK. Thus, Xenopus MAPKK is phosphorylated in mature oocytes by not only an upstream kinase, MAPKKK, but also a downstream kinase, MAPK.

A Xenopus 45 kDa protein has been identified as an immediate upstream factor sufficient for full activation of MAP kinase, and is shown to be capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. In this study, we show that purified 45 kDa protein can phosphorylate a kinase-negative mutant of Xenopus MAP kinase on tyrosine and threonine residues, suggesting that the 45 kDa protein functions as a MAP kinase kinase to activate MAP kinase. We then report the cloning and sequencing of a full-length cDNA encoding this 45 kDa MAP kinase kinase, and show that it is highly homologous to four protein kinases in fission and budding yeasts: byr1, wis1, PBS2 and STE7. These yeast kinases are therefore suggested to function as a direct upstream activator for a presumed MAP kinase homolog in each signal transduction pathway involved in the regulation of cell cycle progression or cellular responses to extracellular signals. Finally, we report bacterial expression of recombinant MAP kinase kinase that can be phosphorylated and activated by Xenopus egg extracts.

The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.

Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.

Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.

MAP kinase is thought to play a pivotal role not only in the growth factor-stimulated signalling pathway but also in the M phase phosphorylation cascade downstream of MPF. MAP kinase is fully active only when both tyrosine and threonine/serine residues are phosphorylated. We have now identified and purified a Xenopus MAP kinase activator from mature oocytes that is able to induce activation and phosphorylation on tyrosine and threonine/serine residues of an inactive form of Xenopus MAP kinase. The Xenopus MAP kinase activator itself is a 45 kDa phosphoprotein and is inactivated by protein phosphatase 2A treatment in vitro. Microinjection of the purified activator into immature oocytes results in immediate activation of MAP kinase. Further experiments using microinjection as well as cell free extracts have shown that Xenopus MAP kinase activator is an intermediate between MPF and MAP kinase. Thus, MAP kinase activator plays a key role in the phosphorylation cascade.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.

Treatment of quiescent rat fibroblastic cells (3Y1) with colchicine, a microtubule-disrupting agent, which could induce the initiation of DNA synthesis [Y. Shinohara, E. Nishida, and H. Sakai (1989) Eur. J. Biochem. 183, 275-280], activated a serine/threonine-specific protein kinase activity in cell extracts that preferentially phosphorylated exogenous microtubule-associated protein 2 (MAP2). Vinblastine treatment also activated the kinase activity, and taxol pretreatment inhibited the colchicine-induced activation of this kinase activity. The detailed biochemical characterization indicated that this microtubule disruption-activated MAP2 kinase was very similar or identical to the mitogen-activated MAP kinase in the substrate specificity and chromatographic behaviors on phosphocellulose, DEAE-cellulose, gel filtration, and phenyl-Sepharose. Pretreatment of the cells with protein synthesis inhibitors did not prevent the MAP2 kinase activation by colchicine. Moreover, phosphatase treatment inactivated the colchicine-activated MAP2 kinase activity. These data suggest that microtubule disruption activates MAP kinase through phosphorylation.

The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A, and is a strong tumor promoter that is not an activator of protein kinase C. Treatment of quiescent cultures of rat fibroblastic 3Y1 cells with okadaic acid induced marked activation of a kinase activity that phosphorylated microtubule-associated protein (MAP) 2 and myelin basic protein, but not histone or casein, in vitro. This activated kinase eluted at approximately 0.15 M NaCl on a DEAE-cellulose column and its apparent molecular mass was determined to be approximately 40 kDa by gel filtration. Detection of the kinase activity in polyacrylamide gels containing substrate proteins after sodium dodecyl sulfate gel electrophoresis revealed that the okadaic-acid-activated kinase activity resided mainly in two closely related polypeptides with apparent molecular mass approximately 40 kDa. The characteristics of this kinase were indistinguishable from those of the mitogen-activated MAP kinase in the same cells. The okadaic-acid-activated MAP kinase was deactivated by protein phosphatase 2A treatment in vitro. These results suggest that MAP kinase is negatively regulated by protein phosphatases 1 and/or 2A in quiescent cells and therefore can be activated by inhibiting these protein phosphatases. Interestingly, the okadaic-acid-induced activation of MAP kinase was transient and epidermal-growth-factor-induced activation was also transient, even in the presence of okadaic acid. These data may imply that protein phosphatases 1 and 2A are not involved in the deactivation of MAP kinase in cells.

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.

We have previously found and characterized a mitogen-activated, serine/threonine-specific protein kinase that specifically phosphorylates microtubule-associated protein 2 (MAP2) in vitro, which we call here MAP2 kinase [Hoshi, M., Nishida, E. & Sakai, H. (1988) J. Biol. Chem. 263, 5396-5401; Hoshi, M., Nishida, E. & Sakai, H. (1989) Eur. J. Biochem. 184, 477-486]. In this study, we have found another serine/threonine-specific protein kinase that is activated by various mitogens. The activated kinase utilized microtubule-associated protein 1B (MAP1B) as the major substrate in vitro, so we tentatively call it MAP1B kinase (M1BK). M1BK was maximally activated 20-30 min after treatment of quiescent rat fibroblastic 3Y1 cells with epidermal growth factor (EGF), while MAP2 kinase was maximally activated within 5-10 min of EGF treatment. The EGF-activated M1BK was eluted at about 0.15 M NaCl on a DEAE-cellulose column, while the activated MAP2 kinase was eluted at about 0.1 M NaCl under the conditions used. The EGF-activated M1BK was eluted as a single peak just after the activated MAP2 kinase on an HPLC gel-filtration column. Histone, casein and ribosomal protein S6 were very poor substrates for the M1BK, while MAP2 and myelin basic protein were moderate substrates. The M1BK activity in cell extracts was inhibited by Ca2+, glycerol 2-phosphate and Zn2+, and slightly enhanced by heparin. These data suggested that M1BK is distinct from previously described mitogen-activated kinases such as MAP2 kinase, casein kinase II and S6 kinase. Pretreatment with cycloheximide or puromycin did not block the M1BK activation by EGF. Furthermore, incubation of the EGF-activated M1BK with acid phosphatase inactivated the kinase activity. Therefore, M1BK may be activated by phosphorylation in EGF-treated cells. In addition to EGF, 12-O-tetradecanoylphorbol 13-acetate, platelet-derived growth factor and insulin-like growth factor-I also induced the activation of M1BK in quiescent cells.