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Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

ABSTRACT: Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors.

SUBMITTER: Coyle SM

PROVIDER: S-EPMC3787944 | biostudies-other | 2013 Aug

REPOSITORIES: biostudies-other

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Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

Coyle Scott M SM Flores Jonathan J Lim Wendell A WA

Cell 20130801 4

Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in ...[more]

PMID: 23953117

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Project description:BackgroundConventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.MethodsBMDM from wild type (WT), MKK3-/-, and MKK6-/- mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.ResultsPre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3-/- BMDM compared with either WT or MKK6-/- cells. IL-10 mRNA expression was modestly reduced in MKK3-/- BMDM but was preserved in MKK6-/- cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3-/- and p38 inhibitor pre-treated cells, but not MKK6-/- cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6-/- cells.ConclusionUnlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA.

Project description:We investigated the relationships of the Cek1 and Cek2 mitogen-activated protein (MAP) kinases and the putative MAP kinase phosphatase Cpp1 in the mating process of Candida albicans Mutants of the CPP1 gene are hyperresponsive to pheromone, generating large halos, high levels of projections, and an increase in pheromone-responsive gene expression. Mating-type-homozygous opaque cells that lack both kinases are sterile, consistent with previous observations, although several lines of evidence show that the two kinases do not simply provide redundant functions in the mating process. Loss of CEK1 reduces mating significantly, to about 0.3% of wild-type strains, and also reduces projection formation and pheromone-mediated gene expression. In contrast, loss of CEK2 has less of an effect, reducing mating to approximately one-third that of the wild-type strain and moderately reducing projection formation but having little influence on the induction of gene expression. However, loss of Cek2 function reduces adaptation to pheromone-mediated arrest. The mutation enhances pheromone response halos to a level similar to that of cpp1 mutants, although the cpp1 mutants are considerably more mating defective than the cek2 mutant. The double cek2 cpp1 mutant shows enhanced responsiveness relative to either single mutant in terms of gene expression and halo formation, suggesting the kinase and phosphatase roles in the adaptation process are independent. Analysis of protein phosphorylation shows that Cek1 undergoes pheromone-mediated phosphorylation of the activation loop, and this phosphorylation is enhanced in cells lacking either the Cpp1 phosphatase or the Cek2 kinase. In addition, Cek1-GFP shows enhanced nuclear localization in response to pheromone treatment. In contrast, Cek2 shows no evidence for pheromone-mediated phosphorylation or pheromone-mediated nuclear localization. Intriguingly, however, deletion of CPP1 enhances both the phosphorylation state and the nuclear localization of Cek2-GFP. Overall, these results identify a complex interaction among the MAP kinases and MAP kinase phosphatase that function in the C. albicans mating pathway.IMPORTANCE MAP kinases and their regulators are critical components of eukaryotic signaling pathways implicated in normal cell behavior as well as abnormal behaviors linked to diseases such as cancer. The mating pathway of the yeast Saccharomyces cerevisiae was central in establishing the MAP kinase paradigm. Here we investigate the mating pathway in a different ascomycete, the fungal pathogen C. albicans In this dimorphic fungus MAP kinases are also implicated in the mating response, with two MAP kinases apparently playing redundant roles in the mating process. This work establishes that while some level of mating can occur in the presence of a single kinase, the Cek1 kinase is most important for mating, while the Cek2 kinase is involved in adaptation to signaling. While both kinases appear to be themselves regulated by dephosphorylation through the action of the Cpp1 phosphatase, this process appears important for mating only in the case of Cek1.

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Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

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Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

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