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Spindle pole bodies function as signal amplifiers in the Mitotic Exit Network.

ABSTRACT: The Mitotic Exit Network (MEN), a budding yeast Ras-like signal transduction cascade, translates nuclear position into a signal to exit from mitosis. Here we describe how scaffolding the MEN onto spindle pole bodies (SPB-centrosome equivalent) allows the MEN to couple the final stages of mitosis to spindle position. Through the quantitative analysis of the localization of MEN components, we determined the relative importance of MEN signaling from the SPB that is delivered into the daughter cell (dSPB) during anaphase and the SPB that remains in the mother cell. Movement of half of the nucleus into the bud during anaphase causes the active form of the MEN GTPase Tem1 to accumulate at the dSPB. In response to Tem1's activity at the dSPB, the MEN kinase cascade, which functions downstream of Tem1, accumulates at both SPBs. This localization to both SPBs serves an important role in promoting efficient exit from mitosis. Cells that harbor only one SPB delay exit from mitosis. We propose that MEN signaling is initiated by Tem1 at the dSPB and that association of the downstream MEN kinases with both SPBs serves to amplify MEN signaling, enabling the timely exit from mitosis.

SUBMITTER: Campbell IW

PROVIDER: S-EPMC7185974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Spindle pole bodies function as signal amplifiers in the Mitotic Exit Network.

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