Proteomics

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Phosphoproteomic analysis of signal transmission in the Mitotic Exit Network


ABSTRACT: In budding yeast, the Mitotic Exit Network (MEN), a GTPase signaling cascade integrates spatial and temporal signals to promote exit from mitosis. This signal integration requires transmission of a signal created on the cytoplasmic face of the spindle pole bodies (SPBs, functional equivalent of the centrosome in yeast) to the nucleolus, where the MEN effector protein Cdc14 resides. In this study, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation by the Polo kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Cdc5 phosphorylates the Cdc14 nucleolar anchor Cfi1/Net1 creating a phospho-binding site for Dbf2-Mob1 which allows Dbf2-Mob1 to phosphorylate Cfi1/Net1 and Cdc14, which activates Cdc14. The mechanisms governing intracellular signal transmission that we uncovered in the MEN – regulated nuclear access and effector activation through priming kinases - may well serve as paradigms for intracellular signal transmission in general. As for phosphoproteomics, our analyses suggest a simple model where Cdc5 and Dbf2-Mob1 phosphorylate Cfi1/Net1 to bring about the release of Cdc14 from its inhibitor. Previous studies have shown that Cfi1/Net1 is a highly phosphorylated protein. To map sites in Cfi1/Net1 that are phosphorylated in a CDC5 or MEN-dependent manner, we performed phosphoproteomics analyses on wild-type anaphase cells and cells in which Cdc5 or Cdc15 were inhibited. We synchronized wild-type, cdc5-as1 and cdc15-as1 cells in G1 with α-factor pheromone and released them into the cell cycle with the corresponding inhibitors. Cultures enriched in anaphase cells as judged by anaphase spindle formation (~70% for wild-type cells and ~95% for cdc5-as1 and cdc15-as1 cells) were collected and processed for a reproducible data-independent acquisition mass spectrometry (DIA-MS) analysis with 7 technical replicates for each sample. Also, 10 out of 12 CDC5-only sites identified in anaphase cells are already phosphorylated in cells arrested in metaphase using the microtubule depolymerizing drug nocodazole with 6 of them were also determined to be CDC5-dependent in metaphase, which is consistent with the finding that Cdc5 is already active in metaphase.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

SUBMITTER: Yansheng Liu  

LAB HEAD: Yansheng Liu

PROVIDER: PXD020369 | Pride | 2021-01-14

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
20190626_Snow_phos_1_f.raw Raw
20190626_Snow_phos_2_f.raw Raw
20190626_Snow_phos_3_f.raw Raw
20190626_Snow_phos_4_f.raw Raw
20190712_Snow_phos_1a.raw Raw
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