Protein phosphatases in MAPK signalling: we keep learning from yeast (original) (raw)
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J Biol Chem, 2009
The prototypic Mitogen Activated Protein Kinase (MAPK) pathway consists of a three-tiered module composed of a MAPK (ERK-1/2), which is activated via phosphorylation by a MAPKK (MEK-1/2), which in turn is phosphorylated by a MAPKKK (Raf). This pathway exists in all eukaryotic organisms and controls fundamental cellular processes such as proliferation, differentiation, migration, survival, and apoptosis [1-3]. Importantly, the cascade arrangement of this module permits integration of a wide range of conserved cellular process, thereby enabling a precise control of the amplitude, kinetics, and duration of ERK-1/2 (ERK) activation. Several studies have documented that it is the ability to modulate these individual parameters of ERK activation that confers signaling specificity to this pathway, in terms of regulating the cellular response [4-7].
Studying the Regulation of MAP Kinase by MAP Kinase Phosphatases In Vitro and in Cell Systems
Methods in Molecular Biology, 2010
Signaling through MAPK pathways involves a network of activating kinases and inactivating phosphatases. While single MAPK kinases account for specific activation of the distinct MAPKs, inactivation of MAPKs by phosphatases involves a wider spectrum of enzymes, with phosphatases from distinct families displaying specificity toward MAPKs. The dual-specificity family of MAPK phosphatases, MKPs, constitutes the major group of MAPK inactivating phosphatases. MKPs are widely expressed, in a tissue-and developmentregulated manner, and the control of their expression and function is crucial for the regulation of MAPK signaling. Here, we present three methods to analyze the regulation of MAPKs by MKPs, using transient and stable-inducible MKP overexpression cell systems and in vitro phosphatase experiments.
Fine regulation of Saccharomyces cerevisiae MAPK pathways by post-translational modifications
Yeast, 2010
Saccharomyces cerevisiae has been widely used as a model eukaryotic organism to elucidate the molecular mechanisms that operate upon activation of signalling pathways. For over two decades, many clues to the regulation of mitogen-activated protein kinase (MAPK) pathways have derived from basic research in yeast. Here we review aspects of MAPK pathway fine-tuning, such as the functional implication of feedback loops or regulatory inputs from other pathways, mediated by post-transcriptional modifications on their components. The impact of recent phosphoproteomic approaches in this particular field is also discussed. Copyright
Molecular Cell, 2001
Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wildtype cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover.
Extracellular signals and scores of phosphatases: All roads lead to MAP kinase
Seminars in Immunology, 2000
MAP kinases function as key signal integration points for a vast number of external stimuli that affect the life and death of cells and are therefore subject to rigourous regulation. Here we review the numerous protein phosphatases that directly counteract MAP kinase activitation. To simplify the complexity, we attempt to integrate the information into a 'sequential phosphatase model' of MAP kinase regulation.
PLoS ONE, 2014
Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved noncanonical docking site in the regulation of the CWI pathway in fungi.
Fungal genetics and biology : FG & B, 2015
The lack of signaling through MAPK pathways leads to a defective cellular response to the corresponding stimulus, but an improper hyperactivation of these routes results in deleterious effects as well. Protein phosphorylation is an activating modification for signal transmission through components of MAPK pathways and thus, protein phosphatases are key negative regulators of these cellular routes by limiting excessive signaling activity. However, in contrast to most of the protein kinases operating in MAPK pathways, protein phosphatases usually exhibit redundancy and promiscuity, which has limited the identification of their function. In order to identify new putative phosphatases operating in Saccharomyces cerevisiae MAPK signaling, we have taken advantage of growth inhibition promoted by overproduction of constitutively active components of the mating and cell wall integrity (CWI) pathways to perform a screen with a collection of 43 protein phosphatases or phosphatase-regulatory p...
Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1
Genetics, 2015
The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAP kinase kinase (MAPKK) known to activate the cell wall integrity (CWI) Slt2 MAP kinase. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal or CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the C...
Signal inhibition by a dynamically-regulated pool of mono-phosphorylated MAPK
Molecular biology of the cell, 2015
Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multi-site phosphorylation, and the addition or removal phosphates can occur in a sequential (processive) or a step-wise (distributive) manner. Here we measured the relative abundance of the mono- and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the mono-phosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5(ND)) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or "synthetic&q...