Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A^Cdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.The eukaryotic cell cycle is a highly coordinated and conserved process. In addition to DNA replication, one of the major requirements for the cell to progress through the cell cycle is the precise duplication of membrane-enclosed organelles and other cellular components before cell division. Knowledge about the mechanisms regulating (membrane) lipid homeostasis during the cell cycle is scarce (1), however several levels of evidence suggest regulation of key enzymes of lipid metabolism in a cell-cycle–dependent manner. The PAH1-encoded phosphatidic acid (PA) phosphatase (Pah1), a key enzyme of triacylglycerol (TG) synthesis that provides the TG precursor diacylglycerol (DG), is phosphorylated and inactivated by the cyclin-dependent kinases Cdc28 and Pho85–Pho80 (2, 3). Kurat et al. showed that Tgl4, next to Tgl3, one of the two major TG lipases in yeast and the ortholog of mammalian ATGL (4, 5), is also phosphorylated by Cdc28. In contrast to Pah1, however, Tgl4 is activated by Cdc28 (6). This inverse regulation of Pah1 and Tgl4 by Cdc28-dependent phosphorylation led to the model by which the TG content oscillates during the cell cycle: On the one hand, TG synthesis serves as a buffer for excess de novo generated fatty acids (FAs), and on the other hand, in times of increased demand—that is, at the onset of bud formation and bud growth—Tgl4-catalyzed lipolysis becomes active to provide TG-derived precursors for membrane lipid synthesis (6).TG and membrane phospholipids share the same intermediates, PA and DG; PA is generated by sequential acylation of glycerol-3-phosphate, reactions that mostly take place in the endoplasmic reticulum (ER) membrane (7). The dephosphorylation of PA to DG by the PAH1-encoded PA phosphatase Pah1 is the major regulator of cellular TG synthesis in yeast (8), similar to its mammalian ortholog, lipin (9). According to this central role, TG content in PAH1-deficient yeast cells is decreased by 70–90%; the source of the residual TG in these mutants is currently unclear. TG synthesis from DG requires one additional acylation step that is catalyzed by the DGA1-encoded acyl-CoA–dependent DG acyltransferase (10, 11) and the phospholipid-dependent acyltransferase, encoded by LRO1 (7). Alternatively, in the presence of the phospholipid precursors, ethanolamine and/or choline, DG may be converted into phospholipids via the Kennedy pathway (7). Thus, net TG synthesis in growing cells is determined by multiple factors, including the availability of FAs, presence of lipid precursors, and the activities of PA phosphatase and the DG acyltransferases. Degradation of TG in yeast is governed by the major lipid droplet (LD)-associated lipases, encoded by TGL3 and TGL4 (4, 12); both enzymes belong to the patatin-domain–containing family of proteins, members of which play a crucial role in lipid homeostasis also in mammals (13). Multiple additional lipases exist in yeast, but their specific function and contribution to TG homeostasis may be restricted to specific growth conditions (7, 14, 15).Absence of lipolysis in mutants lacking TGL3 and TGL4 results in up to threefold elevated levels of TG and reduced levels of phosphatidylcholine and sphingolipids (4, 12, 16, 17), indicating that TG breakdown provides precursors for these lipids or generates some regulatory factors required for their synthesis. The rate of phosphatidylinositol (PI) synthesis after readdition of inositol to inositol-starved cells is reduced by 50% in lipase-deficient cells; the boost of PI synthesis under inositol refeeding conditions is completely abolished if de novo FA synthesis is additionally blocked in the lipase mutants by the inhibitor cerulenin (18). These data clearly demonstrate the requirements for TG breakdown, in addition to de novo FA synthesis, to generate precursors for membrane lipids. As a consequence of defective lipolysis, entry of quiescent cells into vegetative growth is significantly delayed; thus, TG breakdown is particularly important for promoting exit from the stationary phase and entry into the gap1 (G1) phase of the cell cycle (4, 6, 19).Progression through the cell cycle is regulated by specific checkpoint pathways that ensure completion of crucial events and execute a halt under nonconducive conditions. Checkpoint mechanisms slow down or arrest the cell cycle to enable cells to fix damage or to obtain the required metabolites before proceeding and are as such important for the integrity of cell division (20–22). According to this critical function in quality control, mutations in checkpoint genes in mammals have been linked to cancer predisposition and progression. The first discovered cell-cycle checkpoint in Schizosaccharomyces pombe that regulates entry into mitosis is executed by the Wee1 kinase (23, 24), which delays mitosis by phosphorylating and inhibiting cyclin-dependent kinase Cdk1 (25). Conversely, the phosphatase Cdc25 promotes entry into mitosis by removing the inhibitory phosphorylation of Cdk1 (26–28). The budding yeast orthologs of Wee1 and Cdc25 are called Swe1 and Mih1, and their key functions in regulating Cdk1 activity are highly conserved (29, 30). Swe1 phosphorylates Cdk1 (encoded by CDC28 in budding yeast) at the tyrosine 19 residue and inhibits its kinase activity (29, 31, 32); the Mih1 phosphatase removes this inhibitory phosphorylation initiating G2/M cell-cycle progression (26). The Swe1 and Cdk1/Cdc28 kinases operate in an autoregulatory loop in which Swe1 is initially phosphorylated and activated by Cdk1/Cdc28 that is associated with mitotic cyclins; subsequently, activated Swe1 phosphorylates and inhibits Cdk1/Cdc28 (33). The initial phosphorylation of Swe1 is opposed by the protein phosphatase 2A (PP2A) with its catalytic subunits Pph21 or Pph22 and the regulatory subunit Cdc55 (PP2A^Cdc55), which sets a threshold, limiting the activation of Swe1 by Cdk1/Cdc28 in early mitosis (34, 35). Loss of the regulatory subunit Cdc55 leads to hyperactivation of Swe1 (35); after the initial phosphorylation of Swe1 in early mitosis, subsequent phosphorylation events trigger full hyperphosphorylation of Swe1 (33), which leads to its ubiquitin-mediated degradation (36, 37). Of note, regulation of Cdk1/Cdc28 by the G1 cyclin Cln2 plays an important role in actin cytoskeleton polarization and the localized delivery of secretory vesicles, which contribute membrane lipids to the developing bud, thus linking cell surface growth to the cell cycle (38).Despite its proposed role as a gap2 phase (G2) checkpoint regulator, we now show that Swe1 kinase is responsible for the G1/S (Gap1/replication phase) cell-cycle delay in mutants defective in TG lipolysis by phosphorylating Cdk1/Cdc28 at tyrosine 19. Deletion of Swe1 in the tgl3 tgl4 lipase mutant restores normal cell-cycle progression; similarly, supplementation of mutant cells with saturated FAs (myristic acid, palmitic acid) or a precursor of sphingolipid synthesis, phytosphingosine (PHS), suppress the cell-cycle delay in the lipase mutants. These data suggest that Swe1 is a lipid-regulated kinase that is activated in the absence of specific lipids, presumably sphingolipids, and halts G1/S transition by phosphorylating Cdk1/Cdc28 in lipase-deficient cells that exit from the G0 phase of the cell cycle.