In vivo functions of mitogen-activated protein kinases: conclusions from knock-in and knock-out mice

Multicellular organisms achieve intercellular communication by means of signalling molecules whose effect on the target cell is mediated by signal transduction pathways. Such pathways relay, amplify and integrate signals to elicit appropriate biological responses. Protein kinases form crucial intermediate components of numerous signalling pathways. One group of protein kinases, the mitogen-activated protein kinases (MAP kinases) are kinases involved in signalling pathways that respond primarily to mitogens and stress stimuli. In vitro studies revealed that the MAP kinases are implicated in several cellular processes, including cell division, differentiation, cell survival/apoptosis, gene expression, motility and metabolism. As such, dysfunction of specific MAP kinases is associated with diseases such as cancer and immunological disorders. However, the genuine in vivo functions of many MAP kinases remain elusive. Genetically modified mouse models deficient in a specific MAP kinase or expressing a constitutive active or a dominant negative variant of a particular MAP kinase offer valuable tools for elucidating the biological role of these protein kinases. In this review, we focus on the current status of MAP kinase knock-in and knock-out mouse models and their phenotypes. Moreover, examples of the application of MAP kinase transgenic mice for validating therapeutic properties of specific MAP kinase inhibitors, and for investigating the role of MAP kinase in pathogen-host interactions will be discussed.     

Activation of the MAP kinase pathway by the protein kinase raf.

Both MAP kinases and the protein kinase p74raf-1 are activated by many growth factors in a c-ras-dependent manner and by oncogenic p21ras. We were therefore interested in determining the relationship between MAP kinases and raf. The MAP kinase ERK2 is activated by expression of oncogenically activated raf, independently of cellular ras. Overexpressed p74raf-1 potentiates activation of ERK2 by EGF and TPA. MAP kinase kinase inactivated by phosphatase 2A treatment is phosphorylated and reactivated by incubation with p74raf-1 immunoprecipitated from phorbol ester-treated cells. We conclude that raf protein kinase is upstream of MAP kinases and is either a MAP kinase kinase kinase or a MAP kinase kinase kinase kinase.     

MAP kinases: universal multi-purpose signaling tools

MAP (mitogen-activated protein) kinases are serine/threonine protein kinases and mediate intracellular phosphorylation events linking various extracellular signals to different cellular targets. MAP kinase, MAP kinase kinase and MAP kinase kinase kinase are functional protein kinase units that are conserved in several signal transduction pathways in animals and yeasts. Isolation of all three components was also shown in plants and suggests conservation of a protein kinase module in all eukaryotic cells. In plants, MAP kinase modules appear to be involved in ethylene signaling and auxin-induced cell proliferation. Therefore, coupling of different extracellular signals to different physiological responses is mediated by MAP kinase cascades and appears to have evolved from a single prototypical protein kinase module which has been adapted to the specific requirements of different organisms.     

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.     

Characterization of microtubule-associated protein 1-associated protein kinases from rat brain

The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly- -lysine and poly- -arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [-³²P]8-azido ATP indicated that the e65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.     

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.     

Atypical mitogen-activated protein kinases: structure, regulation and functions

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that play a central role in transducing extracellular cues into a variety of intracellular responses ranging from lineage specification to cell division and adaptation. Fourteen MAP kinase genes have been identified in the human genome, which define 7 distinct MAP kinase signaling pathways. MAP kinases can be classified into conventional or atypical enzymes, based on their ability to get phosphorylated and activated by members of the MAP kinase kinase (MAPKK)/MEK family. Conventional MAP kinases comprise ERK1/ERK2, p38s, JNKs, and ERK5, which are all substrates of MAPKKs. Atypical MAP kinases include ERK3/ERK4, NLK and ERK7. Much less is known about the regulation, substrate specificity and physiological functions of atypical MAP kinases.     

Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions. Involvement of the MAP kinases AtMPK3 and AtMPK6

Five Ca(2+)-independent protein kinases were rapidly activated by hypoosmotic stress, moderate or high hyperosmolarity induced by several osmolytes, sucrose, mannitol or NaCl. Three of these kinases, transiently activated by hypoosmolarity, recognised by anti-phosphorylated mitogen-activated protein (MAP) kinase antibodies, sensitive to a MAP kinase inhibitor and inactivated by the action of a tyrosine phosphatase, corresponded to MAP kinases. Using specific antibodies, two of the MAP kinases were identified as AtMPK6 and AtMPK3. The two other protein kinases, durably activated by high hyperosmolarity, did not belong to the MAP kinase family. Activation of AtMPK6 and AtMPK3 by hypoosmolarity depended on upstream protein kinases sensitive to staurosporine and on calcium influx. In contrast, these two transduction steps were not involved in the activation of the two protein kinases activated by high hyperosmolarity.     

Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.     

Crystallization of MAP kinases

X-ray structural studies of MAP kinases and MAP kinase module components are elucidating how kinase activity is regulated and how specificity of signaling is conferred. In the past decade, MAP kinases have been crystallized in their active, phosphorylated forms or low-activity, unphosphorylated forms, as well as in the presence of binding partners such as docking peptides and inhibitors. Crystallization has been achieved via diverse strategies including control of phosphorylation, coding sequence modification, incorporation of tags for purification, and use of a variety of cell-types for protein expression. Recently, interest has been focused on use of crystallography for lead optimization in the development for pharmacological inhibitors on MAP kinases. Further, some success has been gained in crystallizing the MAP kinase activators MAP2Ks and MAP3K kinase domains. This review describes the key methods that have been utilized to crystallize MAP kinases and MAP kinase pathway components.     

Phosphorylation of MAP kinases by MAP/ERK involves multiple regions of MAP kinases.

Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains.     

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Mitogen-activated protein kinases are in vivo transducers of osmosensory signals in fish gill cells

The abundance and activity of three subgroups of mitogen-activated protein (MAP) kinases, the extracellular signal regulated kinase 1 (ERK1), stress-activated protein kinase 1/ Jun N-terminal kinase (SAPK1), and stress-activated protein kinase 2/ p38 (SAPK2), were measured in gill epithelium of the euryhaline teleost Fundulus heteroclitus exposed for 1 h to 4 weeks to hyper- and hyposmotic stress. The abundance of ERK1, SAPK1 and SAPK2 was analyzed by standard Western immunodetection. MAP kinase activity is a function of phosphorylation and was measured using phospho-specific and MAP kinase subgroup-specific antibodies. The abundance of the 63 kDa fish isoform of SAPK2 increases significantly during hyper- but not hyposmotic stress while ERK1 and SAPK1 protein levels remain unchanged during both types of osmotic stress. In contrast to this small effect of osmotic stress on MAP kinase abundance, the activity of all MAP kinases decreases significantly in response to hyperosmotic stress and increases significantly during hyposmotic stress. These results demonstrate for the first time that the activity of all major MAP kinases is osmoregulated in gill epithelium of euryhaline fish. Based on these results we conclude that MAP kinases are important components of salinity adaptation and participate in osmosensory signaling pathways in gill epithelium of euryhaline fishes.     

cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates.

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.     

Expression and activation of RAS and mitogen-activated protein kinases in macrophages treated in vitro with cisplatin: regulation by kinases, phosphatases and Ca2+/calmodulin.

Cisplatin (cis-dichlorodiammineplatinum II), a potent antitumour compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the exact mechanism by which cisplatin activates these cells is poorly characterized and attempts are being made to understand this mechanism. Previous studies from this laboratory have shown that Lyn, a protein tyrosine kinase of the src family, and nuclear factor (NF)-kappaB are involved in cisplatin-induced macrophage activation. Recent studies suggest that the RAS and mitogen-activated protein (MAP) kinases function as a connecting link between activated lyn and NF-kB, which raises the possibility of their involvement in cisplatin-induced macrophage activation. Therefore, this study was undertaken to investigate the effect of cisplatin treatment on the expression/activation of RAS (a low molecular weight GTP-binding protein) and MAP kinases in murine peritoneal macrophages. The underlying mechanism of expression/activation of RAS and MAP kinases in cisplatin-treated macrophages was also investigated. Immunoblotting and immune-complex kinase assays revealed that cisplatin treatment of macrophages leads to increased expression/activation of RAS and MAP kinases, with optimal expression/activation at 15 min of treatment. Using a battery of specific inhibitor/modulators of different signalling molecules, this study shows that expression and activation of MAP kinases are two unrelated processes. It was also observed that kinase (protein tyrosine and protein kinase C) inhibitor and Ca2+/calmodulin antagonist inhibit expression/activation of RAS/MAP kinases in macrophages, whereas phosphatases (protein tyrosine and serine/threonine) inhibitor up-regulate these kinases.     

Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.     

MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response?

Hypoosmotic stress activates a phosphorylation-dependent oxidative burst. In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like. Apigenin, a MAP kinase and cyclin-dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst ( Cazaléet al. 1998 ; Plant Physiol. 116, 659–669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6-DMAP. However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst. Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.     

Production of active recombinant mitogen-activated protein kinases through transient transfection of 293T cells

Mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP kinases has been shown to participate in a variety of human diseases which include cancer, septic shock, rheumatoid arthritis, diabetes, and cardiovascular diseases. Active MAP kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic threonine and tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP kinases. We cloned JNK1, p38, and p38-regulated MAP kinase-activated protein kinase-2 into the mammalian expression vector pEBG, and expressed these protein kinases as glutathione S-transferase fusion proteins in human embryonic kidney 293T cells through transient transfection. The protein kinases were activated in vivo through treating the transfected cells with sodium arsenite and affinity-purified using glutathione-Sepharose beads. The enzymatic activities of these protein kinases were demonstrated by Western blot analysis and in vitro kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable reagents, and could be very valuable for proteomic studies.