Cell cycle phosphorylation of mitotic exit network (MEN) proteins

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.Key words: in vivo phosphorylation, yeast centrosome, mitotic exit network (MEN), cell cycle, protein kinase, Cdk (cyclin-dependent kinase)/Cdc28, Plk1 (polo-like kinase)/Cdc5Reversible protein phosphorylation leads to changes in targeting, structure and stability of proteins and is used widely to modulate biochemical reactions in the cell. We are interested in phosphoregulation of centrosome duplication and function in the yeast Saccharomyces cerevisiae. Centrosomes nucleate microtubules and, upon duplication during the cell cycle, form the two poles of the bipolar mitotic spindle used to segregate replicated chromosomes into the two daughter cells. Timing and spatial cues are highly regulated to ensure that elongation of the mitotic spindle and separation of sister chromatids occur prior to progression into late telophase and initiation of mitotic exit. The mitotic exit network (MEN) regulates this timing through a complex signaling cascade activated at the centrosome that triggers the end of mitosis, ultimately through mitotic cyclin-dependent kinase (Cdk) inactivation (reviewed in ref. ¹).The major components of the MEN pathway (Fig. 1) are a Ras-like GTPase (Tem1), an activator (Lte1) with homology to nucleotide exchange factors, a GTPase-activating protein (GAP) complex (Bfa1/Bub2), several protein kinases [Cdc5 (Plk1 in humans), Cdc15 and Dbf2/Mob1] and Cdc14 phosphatase (reviewed in ref. ^2–5). Tem1 initiates the signal for the MEN pathway when switched to a GTP-active state. Prior to activation at anaphase, it is held at the centrosome in an inactive GDP-bound state by an inhibiting GAP complex, Bfa1/Bub2.⁶ The Bfa1/Bub2 complex and the inactive Tem1 are localized at the mother centrosome destined to move into the budded cell upon chromosome segregation, whereas the activator Lte1 is localized at the tip of the budded cell. These separate localizations ensure that Lte1 and Tem1 only interact in late anaphase, when the mitotic spindle elongates.^7,8 Lte1 has been thought to activate Tem1 as a nucleotide exchange factor, although more recent evidence suggests that it may instead affect Bfa1 localization.⁹ In addition, full activation of Tem1 is achieved through Cdc5 phosphorylation of the negative regulator Bfa1 ¹⁰ and potentially through phosphorylation of Lte1. GTP-bound Tem1 is then able to recruit Cdc15 to the centrosome, allowing for Dbf2 activation.³ The final step in the MEN pathway is release of Cdc14 from the nucleolus, which is at least partially due to phosphorylation by Dbf2¹¹ an leads to mitotic cyclin degradation and inactivation of the mitotic kinase.²Open in a separate windowFigure 1Schematic representation of the MEN proteins and pathway. MEN protein localization is shown within a metaphase cell when mitotic exit is inhibited and in a late anaphase cell when mitotic exit is initiated. Primary inhibition and activation events are described below the cells.Recently, we performed a large-scale analysis of phosphorylation sites on the 18 core yeast centrosomal proteins present in enriched centrosomal preparations.¹² In total, we mapped 297 sites on 17 of the 18 proteins and described their cell cycle regulation, levels of conservation and demonstrated defects in centrosome assembly and function resulting from mutating selected sites. MEN proteins were also identified in the centrosome preparations. This was expected, because Nud1, one of the 18 core centrosome components, is known to recruit several MEN proteins to the centrosome¹³ as part of its function in mitotic exit.^14,15 As phosphorylation is essential to several steps in the MEN pathway, beginning with recruitment of Bfa1/Bub2 by phosphorylated Nud1,¹⁵ we were interested in mapping in vivo phosphorylation sites on the MEN proteins associated with centrosomes and identifying when they occur during the cell cycle.We combined centrosome enrichment with mass spectrometry analysis to examine phosphorylation from asynchronously growing cells.¹² Centrosomes were also prepared from cells arrested in G₁ and mitosis¹² to monitor potentially cell cycle-regulated sites. We obtained significant coverage of a number of the MEN proteins, several of which have human homologs (​and33, column 1), of which eight sites lie within Cdk/Cdc28 motifs [S/T(P)], (23 Mob1 and Dbf2 are known phosphoproteins²⁴ for which we observed peptide coverage but no phosphorylation. Surprisingly, we did not detect phosphorylation on Bub2 despite the high peptide coverage; it is possible that the mitotic centrosome preparations (using a Cdc20 depletion protocol) affect the phosphorylation state of Bub2, as Bub2 is required for mitotic exit arrest in cdc20 mutants.²⁵ Additionally, specific phosphorylation sites have not been mapped on Bub2, suggesting that modifications on this protein may be difficult to observe by mass spectrometry. Lte1 does not localize to the centrosome, and we did not recover Lte1 peptides in our preparations. Many phosphorylation events on MEN proteins were observed in mitotic centrosomal preparations, most likely in preparation for their subsequent role in exit from mitosis (