Regulation of MAP kinases by MAP kinase phosphatases

MAP kinase phosphatases (MKPs) catalyze dephosphorylation of activated MAP kinase (MAPK) molecules and deactivate them. Therefore, MKPs play an important role in determining the magnitude and duration of MAPK activities. MKPs constitute a structurally distinct family of dual-specificity phosphatases. The MKP family members share the sequence homology and the preference for MAPK molecules, but they are different in substrate specificity among MAPK molecules, tissue distribution, subcellular localization and inducibility by extracellular stimuli. Our understanding of their protein structure, substrate recognition mechanisms, and regulatory mechanisms of the enzymatic activity has greatly increased over the past few years. Furthermore, although there are a number of MKPs, that have similar substrate specificities, non-redundant roles of MKPs have begun to be identified. Here we focus on recent findings regarding regulation and function of the MKP family members as physiological regulators of MAPK signaling.     

Regulation of melanosome movement by MAP kinase

Our objectives were to further characterize the signaling pathways in melatonin-induced aggregation in Xenopus melanophores, specifically to investigate a possible role of mitogen-activated protein kinase (MAPK). By Western blotting we found that melatonin activates MAPK, which precedes melanosome aggregation measured in a microplate reader. Activation of MAPK, tyrosine phosphorylation of a previously described 280-kDa protein, and melanosome aggregation are sensitive to PD98059, a selective inhibitor of MAPK kinase. The MAPK activation is also decreased by the adenylate cyclase stimulant forskolin. In summary, we found that MAPK is activated during melatonin-induced melanosome aggregation. Activation was decreased by an inhibitor of MAPK kinase, and by forskolin. In addition to inhibition of cyclic adenosine 3',5'-monophosphate (cAMP), reduction in protein kinase A activity (PKA), and activation of protein phosphatase 2A, we suggest that melatonin receptors activate the MAPK cascade and tyrosine phosphorylation of the 280-kDa protein. Although the cAMP/PKA signaling pathway is the most prominent, our data suggest that simultaneous activation of the MAPK cascade is of importance to obtain a completely aggregated state. This new regulatory mechanism of organelle transport by the MAPK cascade might be important in other eukaryotic cells.     

Protein scaffolds in MAP kinase signalling

The mitogen-activated protein kinase (MAPK) pathway allows cells to interpret external signals and respond in an appropriate way. Diverse cellular functions, ranging from differentiation and proliferation to migration and inflammation, are regulated by MAPK signalling. Therefore, cells have developed mechanisms by which this single pathway modulates numerous cellular responses from a wide range of activating factors. This specificity is achieved by several mechanisms, including temporal and spatial control of MAPK signalling components. Key to this control are protein scaffolds, which are multidomain proteins that interact with components of the MAPK cascade in order to assemble signalling complexes. Studies conducted on different scaffolds, in different biological systems, have shown that scaffolds exert substantial control over MAPK signalling, influencing the signal intensity, time course and, importantly, the cellular responses. Protein scaffolds, therefore, are integral elements to the modulation of the MAPK network in fundamental physiological processes.     

Regulation of MAP kinase by the BMP-4/TAK1 pathway in Xenopus ectoderm

Bone morphogenetic protein-4 (BMP-4) induces epidermis and represses neural fate in Xenopus ectoderm. Our previous findings implicate p42 Erk MAP kinase (MAPK) in the response to neural induction. We have examined the effects of BMP-4 on MAPK activity in gastrula ectoderm. Expression of a dominant negative BMP-4 receptor resulted in a 4.5-fold elevation in MAPK activity in midgastrula ectoderm. MAPK activity was reduced in ectoderm expressing a constitutively active BMP-4 receptor, or ectoderm treated with BMP-4 protein in the presence or absence of cycloheximide. Overexpression of TAK1 led to a reduction in MAPK activity in early gastrula ectoderm. The inhibitory effects of TAK1 could be reversed by 1 microM SB 203580, a p38 inhibitor. Treatment of isolated ectoderm with SB 203580 led to expression of otx2, NCAM, and noggin. Western blot analyses indicated that the BMP-4 pathway does not activate JNKs in ectoderm. Our findings indicate that BMP-4 inhibits ectodermal MAPK activity through a TAK1/p38-type pathway. MAPK has been shown to inactivate Smad1. Thus, our results suggest that BMP-4 and MAPK pathways are mutually antagonistic in Xenopus ectoderm, and that interactions between these pathways may govern the choice between epidermal and neural fate.     

Recent advances in plant MAP kinase signalling

Mitogen activated protein kinases (MAPK) are important mediators in signal transmission, connecting the perception of external stimuli to cellular responses. MAPK cascades are involved in signalling various biotic and abiotic stresses, like wounding and pathogen infection, temperature stress or drought, but are also involved in mediating the action of some plant hormones, such as ethylene and auxin. Moreover, MAPKs have been implicated in cell cycle and developmental processes. In Arabidopsis mutant screens and in vivo assays several components of plant MAPK cascades have been identified. This review gives an update of recent advances in plant MAPK signalling and discusses the emerging mechanisms of some selected MAPK pathways.     

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.     

Deciphering the MAP kinase pathway

MAP kinases (MAPK) are serine/threonine kinases which are activated by a dual phosphorylation on threonine and tyrosine residues. Their specific upstream activators, called MAP kinase kinases (MAPKK), constitute a new family of dual-specific threonine/tyrosine kinases, which in turn are activated by upstream MAP kinase kinase kinases (MAPKKK). These three kinase families are successively stimulated in a cascade of activation described in various species such as mammals, frog, fly, worm or yeast.In mammals, the MAP kinase module lies on the signaling pathway triggered by numerous agonists such as growth factors, hormones, lymphokines, tumor promoters, stress factors, etc. Targets of MAP kinase have been characterize tin all subcellular compartments. In yeast, genetic epistasis helped to characterize the presence of several MAP kinase modules in the same system. By complementation tests, the relationships existing between phylogenetically distant members of each kinase family have been described. The roles of the MAP kinase cascade have been analyzed by engineering various mutations in the kinases of the module. The MAP kinase cascade has thus been implicated in higher eukaryotes in cell growth, cell fate and differentiation, and in low eukaryotes, in conjugation, osmotic stress, cell wall constrct and mitosis.     

Involvement of the mitogen-activated protein (MAP) kinase signalling pathway in host cell invasion by Toxoplasma gondii

Little is known about signalling in Toxoplasma gondii, but it is likely that protein kinases might play a key role in the parasite proliferation, differentiation and probably invasion. We previously characterized Mitogen-Activated Protein (MAP) kinases in T. gondii lysates. In this study, cultured cells were tested for their susceptibility to Toxoplasma gondii infection after tachyzoite pretreatment with drugs interfering with MAP kinase activation pathways. Protein kinases inhibitors, i.e. genistein, RO31-8220 and PD098059, reduced tachyzoite infectivity by 38 +/- 4.5%, 85.5 +/- 9% and 56 +/- 10%, respectively. Conversely, protein kinases activators, i.e. bombesin and PMA, markedly increased infectivity (by 202 +/- 37% and 258 +/- 14%, respectively). These results suggest that signalling pathways involving PKC and MAP kinases play a role in host cell invasion by Toxoplasma.     

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.     

Regulation of the kinase activity of the MIK GCK-like MAP4K by alternative splicing

Alternative splicing of introns is essential to ensure the complexity of mammalian genome functions. In particular, the generation of a high number of different isoforms by alternative splicing is an important characteristic of genes coding for signalling proteins such as mitogen activated protein kinases (MAPKs). This is thought to allow these proteins to transduce multiple stimuli in a highly regulated manner. Plant genes are also subjected to alternative splicing. Nevertheless, clear examples of the functional consequences of this phenomenon are still scarce in plants. MIK is a maize gene coding for a GCK-like MAP4K that can be activated by interaction with maize atypical receptor kinase (MARK), an atypical receptor kinase. Here we show that MIK is subjected to alternative splicing. Expression of MIK leads to, at least, 4 different mature mRNAs that accumulate with particular expression profiles during maize development. Our results show that the polypeptides encoded by the different MIK mRNAs display different kinase activity and are differentially activated by interaction with the MARK receptor. Two MIK isoforms display constitutive kinase activity, one isoform is inactive but can be activated by MARK, and the fourth MIK isoform is inactive and cannot be activated by MARK. Our results constitute a clear example of the biochemical consequences of alternative splicing in plants. The selective conservation during evolution of the intron–exon structure of the region coding for the regulator domain of MIK, as well as the maintenance in maize, rice and Arabidopsis of the alternative splicing of some of these introns, are strong indications of its functional importance.     

Emerging MAP kinase pathways in plant stress signalling

Mitogen-activated protein kinase (MAPK) pathways transfer information from sensors to cellular responses in all eukaryotes. A surprisingly large number of genes encoding MAPK pathway components have been uncovered by analysing model plant genomes, suggesting that MAPK cascades are abundant players of signal transduction. Recent investigations have confirmed major roles of defined MAPK pathways in development, cell proliferation and hormone physiology, as well as in biotic and abiotic stress signalling. Latest insights and findings are discussed in the context of novel MAPK pathways in plant stress signalling.     

Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration

Recent research carried out in our laboratory has shown that IGF-1, TGF-beta1, and insulin were able to strongly stimulate myoblast migration by increasing milli-calpain expression and activity. However, the signalling pathways involved in these phenomena remain unknown. The aim of this study was to identify the signalling pathway(s) responsible for the effects of IGF-1, TGF-beta1, and insulin on myoblast migration and on milli-calpain expression and activity. For this purpose, wound healing assays were carried out in the presence of growth factors with or without specific inhibitors of ERK/MAP kinase and PI3K/Akt pathways. The results clearly showed that the inhibition of the ERK/MAP kinase pathway prevents the effects of growth factors on myoblast migration. Secondly, the expression and the activity of milli-calpain were studied in cells treated with growth factor, alone or with ERK/MAP kinase inhibitor. The results demonstrated that the up-regulation of milli-calpain expression and activity was mediated by the ERK/MAP kinase pathway. Finally, the possible implication of MyoD and myogenin, myogenic regulatory factors able to regulate milli-calpain expression, was studied. Taken together our results clearly showed that the ERK/MAP kinase signalling pathway is responsible for the effects of the three growth factors on myoblast migration and on milli-calpain expression and activity. On the opposite, the PI3K/Akt signalling pathway, MyoD and myogenin seem to be not implicated in these phenomena.     

Acetylcholine muscarinic receptor regulation of the Ras/Raf/MAP kinase pathway

Acetylcholine muscarinic m1 receptors and m2 receptors are predominantly coupled to the heterotrimeric G proteins Gq, 11 and Gi, respectively. Stimulation of the m1 and m2 receptors in different cell types activate the Ras/Raf/MAP kinase pathway. The ability of the m1 receptor to activate the MAP kinase pathway is dependent on the isoforms of adenylyl cyclase expressed in specific cell types. Specific adenylyl cyclases respond to different signals, including calcium and protein kinase C, with increased cAMP synthesis resulting in protein kinase A activation. Stimulation of protein kinase A inhibits Raf and subsequent MAP kinase activation by G protein-coupled receptors and growth factor receptor tyrosine kinases. G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated response pathways.     

Regulation of innate immune response by MAP kinase phosphatase-1

Mitogen-activated protein (MAP) kinase cascades are signal transduction pathways that play pivotal regulatory roles in the biosynthesis of pro-inflammatory cytokines. MAP kinase phosphatase (MKP)-1, an archetypal member of the MKP family, is essential for the dephosphorylation/deactivation of MAP kinases p38 and JNK. Earlier studies conducted using cultured immortalized macrophages provided compelling evidence indicating that MKP-1 deactivates p38 and JNK, thereby limiting pro-inflammatory cytokine biosynthesis in innate immune cells exposed to microbial components. Recent studies employing MKP-1 knockout mice have confirmed the central function of MKP-1 in the feedback control of p38 and JNK activity as well as the crucial physiological function of MKP-1 as a negative regulator of the synthesis of pro-inflammatory cytokines in vivo. MKP-1 was shown to be a major feedback regulator of the innate immune response and to play a critical role in preventing septic shock and multi-organ dysfunction during pathogenic infection. In this review, we will update the studies on the biochemical properties and the regulation of MKP-1, and summarize our understanding on the physiological function of this key phosphatase in the innate immune response.     

Regulation of bidirectional melanosome transport by organelle bound MAP kinase

Regulation of intracellular transport plays a role in a number of processes, including mitosis, determination of cell polarity, and neuronal growth. In Xenopus melanophores, transport of melanosomes toward the cell center is triggered by melatonin, whereas their dispersion throughout the cytoplasm is triggered by melanocyte-stimulating hormone (MSH), with both of these processes mediated by cAMP-dependent protein kinase A (PKA) activity [1, 2]. Recently, the ERK (extracellular signal-regulated kinase) pathway has been implicated in regulating organelle transport and signaling downstream of melatonin receptor [3, 4]. Here, we directly demonstrate that melanosome transport is regulated by ERK signaling. Inhibition of ERK signaling by the MEK (MAPK/ERK kinase) inhibitor U0126 blocks bidirectional melanosome transport along microtubules, and stimulation of ERK by constitutively active MEK1/2 stimulates transport. These effects are specific because perturbation of ERK signaling has no effect on the movement of lysosomes, organelles related to melanosomes [5]. Biochemical analysis demonstrates that MEK and ERK are present on melanosomes and transiently activated by melatonin. Furthermore, this activation correlates with an increase in melanosome transport. Finally, direct inhibition of PKA transiently activates ERK, demonstrating that ERK acts downstream of PKA. We propose that signaling of organelle bound ERK is a key pathway that regulates bidirectional, microtubule-based melanosome transport.     

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart’s insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 μg/mg (~1 mole insulin/mole (α₂β₂) receptor) and a curvilinear Scatchard plot with a high affinity K_D for insulin binding of ~4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg²⁺ (half-maximum response at ~5.6–10.6 nM and ~8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg²⁺ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor’s tyrosine kinase activity by the intracellular free Mg²⁺ concentration and the polyamines in the absence and presence of insulin.     

Regulation of cellular functions by the ERK5 signalling pathway

Extracellular-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase (MAPK) regulated by a wide range of mitogens and cellular stresses. Since its cloning in 1995, the lack of biological tools, including antibodies and specific inhibitors, have made it one of the least studied MAPK subfamilies. The discovery that ERK5 was an important contributor to cell survival mechanisms has increased interest in this signalling pathway. The ability of inhibitors of the classical MAPK (ERK1/2) cascade to block ERK5 activation suggested that ERK5 might regulate some cellular functions originally attributed to ERK1/2. For example, ERK5 is suspected to mediate the effects of numerous oncogenes. A link between abnormal levels of ERK5 expression and cancers was established by the analysis of human tumours. Recently, the targeted deletions of the erk5 and the mek5 genes in mice have provided genetic evidence that the ERK5 cascade is a non-redundant signalling pathway essential for normal cardiovascular development. The analysis of genetically modified mice in which the erk5 gene can be specifically deleted in certain tissues is shedding light into the physiological function of the ERK5 pathway during development and pathogenesis.