Phosphorylation of the RGS Protein Sst2 by the MAP Kinase Fus3 and Use of Sst2 as a Model To Analyze Determinants of Substrate Sequence Specificity

Parnell, Stephen C.; Marotti, Louis A.; Kiang, Lee; Torres, Matthew P.; Borchers, Christoph H.; Dohlman, Henrik G.

doi:10.1021/bi0503091

Cited by 24 publications

(31 citation statements)

References 35 publications

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“…To identify potential direct MAK-2 targets, we inspected the 96 phosphopeptides that showed reduced abundance in the 1NM-PP1 treated mak-2 Q100G germlings for MAPK consensus phosphorylation sites (P-X-S*/T*-P) [36] , [37] . Nine proteins were identified, of which five were annotated as hypothetical proteins.…”

Section: Resultsmentioning

confidence: 99%

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

et al. 2014

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Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.

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Section: Resultsmentioning

confidence: 99%

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

et al. 2014

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“…Here, we further analyzed this phosphoproteomic dataset to identify other targets of MAK-2 and potential members of the HAM-5/MAK-2 complex. Of these 3200 phosphopeptides, 217 phosphopeptides representing 144 proteins have a phosphorylated MAPK consensus site (P-X-S/T-P) (Parnell et al 2005;Mok et al 2010) (File S1). Twentyeight of these 217 peptides showed a lower abundance after MAK-2 kinase inhibition (.1.25-fold change), (Table S4), but because of variability in samples, the difference was not statistically significant (P , 0.05, Student's t-test).…”

Section: Ham-5 Localization Is Not Disrupted In Other Fusion Mutantsmentioning

confidence: 99%

Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14

et al. 2016

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In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5.KEYWORDS cell fusion; Neurospora crassa; MAP kinase signaling; protein complexes; chemotropism F USION between genetically identical cells exists in many organisms and plays an important role in different developmental processes, for example, myoblast fusion during the formation of muscle tissue, macrophage fusion, or fusion of trophoblasts in placental development (Chen and Olson 2005;Aguilar et al. 2013). In fungi, vegetative fusion enables a fungal colony to share resources and has implications for fitness and virulence (Craven et al. 2008;Fricker et al. 2009;Eaton et al. 2011;Richard et al. 2012;Simonin et al. 2012;Roper et al. 2013;Chagnon 2014). In the filamentous ascomycete fungus, Neurospora crassa, genetically identical germinated asexual spores (germlings) undergo fusion to form a syncytium that is an interconnected network of fused cells Leeder et al. 2013;Herzog et al. 2015). An important pathway required for both germling and hyphal fusion is a conserved mitogen-activated protein kinase (MAPK) cascade that includes the MAPK kinase kinase NRC-1, the MAPK kinase MEK-2, the MAPK MAK-2, and the scaffold protein, HAM-5 (Pandey et al. 2004;Dettmann et al. 2012Dettm...

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“…An advantage of pheromone supersensitivity induced by SST2 gene disruption was confirmed by both EGFP fluorescence with Fus1p expression and growth arrest with Far1p phosphorylation. The principal negative regulator of the pathway is Sst2p, which is a GTPase‐activating protein for Gpa1p and belongs to the regulator of G protein signaling (RGS) family, and yeast cells lacking functional Sst2p exhibit a hypersensitivity in the presence of a pheromone (6, 16, 21). As shown in concentration−response curves (Figure 5A), IMGH‐5 lacking Sst2p was 250−1000 times supersensitized to the α‐factor compared with IMGH‐1 when grown in a liquid medium.…”

Section: Discussionmentioning

confidence: 99%

Quantitative and Dynamic Analyses of G Protein-Coupled Receptor Signaling in Yeast Using Fus1, Enhanced Green Fluorescence Protein (EGFP), and His3 Fusion Protein

et al. 2006

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The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.

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Phosphorylation of the RGS Protein Sst2 by the MAP Kinase Fus3 and Use of Sst2 as a Model To Analyze Determinants of Substrate Sequence Specificity

Cited by 24 publications

References 35 publications

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14

Quantitative and Dynamic Analyses of G Protein-Coupled Receptor Signaling in Yeast Using Fus1, Enhanced Green Fluorescence Protein (EGFP), and His3 Fusion Protein

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