Dataset Information

CDK and MAPK collaborate to regulate signaling dynamics via multi-site phosphorylation of the scaffold protein Ste5

ABSTRACT: CDKs and MAPKs phosphorylate similar sites yet generally have distinct functions/substrates. We report here an unanticipated system of cooperative regulation by CDK and MAPK, involving collaborative multi-site phosphorylation of a single substrate. The budding yeast protein Ste5 is a signaling scaffold for the pheromone-activated G1 arrest pathway. Upon cell cycle entry, CDK activity inhibits Ste5 via phosphorylation at numerous sites flanking its membrane-binding domain. Ste5 is also regulated by negative feedback from the pathway MAPK, though the mechanism was unknown. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell cycle stages. Surprisingly, at stages where we expected Ste5 recruitment would be blocked, we observed transient recruitment followed by a steep decline, depending on both CDK and MAPK activities. The collective results of mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK target shared sites, and that the substrate is most extensively phosphorylated when both kinases are active and able to bind their respective docking sites on Ste5. This collaborative phosphorylation can diversify regulatory options, ranging from mild tuning to strong blocking, and can yield distinct patterns of regulatory dynamics at different cell cycle stages.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Haploid Cell

SUBMITTER: Wolfgang Reiter

LAB HEAD: Wolfgang Reiter

PROVIDER: PXD006154 | Pride | 2018-03-12

REPOSITORIES: Pride

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Publications

CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5.

Repetto María Victoria MV Winters Matthew J MJ Bush Alan A Reiter Wolfgang W Hollenstein David Maria DM Ammerer Gustav G Pryciak Peter M PM Colman-Lerner Alejandro A

Molecular cell 20180301 6

We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at diffe ...[more]

PMID: 29547722

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Project description:Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress. In this study we sought to analyze the chromatin structural changes that take place at sites of DNA replication. To this end, we obtained yeast cell populations synchronously undergoing DNA replication by M-NM-1-factor G1 arrest and release. In order to analyze chromatin structure at sites of DNA replication we first mapped the genomic locations undergoing DNA replication to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Two complementary approaches were taken: (1) we mapped fork positions by chromatin immunoprecipitation (ChIP) of a FLAG-tagged DNA polymerase 1 (Pol1), a replication fork component and (2) we mapped the sites of active DNA synthesis by generating DNA copy number profiles. We then analyzed chromatin structure at the sites of DNA replication by Micrococcal nuclease mononucleosome mapping and by generating histone H3 density maps. We further generated a Normalized Chromatin Accessibility to MNase (NCAM) signal by normalizing MNase mononucleosome signal for histone H3 density. NCAM signal represents a measure of nucleosome accessibility to MNase that is normalized for nucleosome content. These three approaches allowed us to assess potential changes in nucleosome positioning, nucleosome density, and nucleosome accessibility during DNA replication. We searched for changes in chromatin structure by comparing, during S phase, regions undergoing DNA replication to those not yet replicated, and also by comparing the same region before replication (not replicated, G1 arrested control) and during DNA replication in S phase. We typically harvested S phase cells at 30 or 60 minutes after release from G1 arrest. Experimental conditions included releasing cells into rich media at 24M-BM-0C or into rich media containing 200 mM Hydroxyurea (HU). Both conditions slowed down replication fork rate and made these experiments feasible. For each strain, samples for the different experiments (Chromatin for Pol1 and H3 ChIP, in vivo MNase digestion, and DNA for DNA copy number profiles) were harvested simultaneously for each time point. Therefore, comparisons between time points for each strain must be made using samples from the same replicate experiment. Our analysis included WT cells, as well as S phase checkpoint mutants (M-NM-^Tmec1 M-NM-^Tsml1 and mec1-100 M-NM-^Tsml1), as well as control strains (M-NM-^Tsml1).

Dataset Information

CDK and MAPK collaborate to regulate signaling dynamics via multi-site phosphorylation of the scaffold protein Ste5

Publications

CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5.

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