Whi5 and Stb1 define redundant pathways through which the G1 cyclin Cln3 promotes cell division

In the budding yeast S. cerevisiae, commitment to cell division, Start, is promoted by a trio of G1 cyclins, Cln1, Cln2, and Cln3, that activate the CDK kinase Cdc28. The active kinases somehow activate two transcription factors, SBF and MBF, leading to induction of about 100 genes for budding, DNA synthesis, and other early cell cycle processes. Activation of the transcription factors is opposed by a repressive protein called Whi5, and also by a second repressive protein called Stb1. Both Whi5 and Stb1 contain many potential sites for phosphorylation by CDK kinase, and is thought that relief of transcriptional repression involves the phosphorylation of Whi5 and Stb1 by CDK. Phosphorylation site mutants have been studied for Whi5, but not for Stb1. Here, we create phosphorylation site mutants of Stb1, and combine them with site mutants of Whi5. We find that the G1 cyclin Cln3 activates cell cycle transcription effectively when at least one of these proteins has its phosphorylation sites. However, when both Whi5 and Stb1 simultaneously lack all consensus phosphorylation sites, Cln3 is unable, or almost unable, to induce any gene expression, or any advancement of Start. Thus the G1 cyclin signaling pathway to Start has a requirement for CDK phosphorylation sites on either Whi5 or Stb1.

Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 is thought to initiate cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that control cell growth and size. Together, the data are consistent with models in which Cln3 is a crucial link between cell growth and the cell cycle.

Localized phosphorylation of RNA Polymerase II by G1 cyclin-Cdk promotes cell cycle entry

The cell cycle is thought to be initiated by cyclin-dependent kinases (Cdk) inactivating transcriptional inhibitors of cell cycle gene-expression. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate Whi5, thereby releasing the transcription factor SBF and committing cells to division. Here, we report that Cln3-Cdk1 does not phosphorylate Whi5, but instead phosphorylates the RNA Polymerase II subunit Rpb1 C-terminal domain (CTD) on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters and Cln3 function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to activate transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these processes.

Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 initiates cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that play roles in control of cell growth and size. Together, the data are consistent with models in which Cln3 serves as the crucial link between the cell cycle and signals that control cell growth and size.

Capsaicin Targets tNOX (ENOX2) to Inhibit G1 Cyclin/CDK Complex, as Assessed by the Cellular Thermal Shift Assay (CETSA)

Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed in cancer but not in non-transformed cells. This attenuates cancer cell growth by inducing apoptosis. The capsaicin-mediated inhibition of tNOX was recently shown to prolong the cell cycle. However, the molecular events underlying this regulation have not yet been investigated. In the present study, we used a cellular thermal shift assay (CETSA) to detect “target engagement” of capsaicin and its consequent impact on cell cycle progression. Our results indicated that capsaicin engaged with tNOX and triggered the proteasomal degradation of tNOX, which leads to the inhibition of NAD+-dependent SIRT1 deacetylase. Ultimately, the acetylation levels of c-Myc and p53 were enhanced, which suppressed the activation of G1 cyclin/Cyclin-dependent kinase complexes and triggered cell cycle arrest in cancer cells. The results obtained when tNOX was overexpressed in non-cancer cells validated its importance in cell cycle progression. These findings provide the first molecular insights into the regulatory role of tNOX and the anti-proliferative property of capsaicin in regulating the cell cycle of bladder cancer cells.

Proteostasis collapse, a hallmark of aging, hinders the chaperone-Start network and arrests cells in G1

Loss of proteostasis and cellular senescence are key hallmarks of aging, but direct cause-effect relationships are not well understood. We show that most yeast cells arrest in G1 before death with low nuclear levels of Cln3, a key G1 cyclin extremely sensitive to chaperone status. Chaperone availability is seriously compromised in aged cells, and the G1 arrest coincides with massive aggregation of a metastable chaperone-activity reporter. Moreover, G1-cyclin overexpression increases lifespan in a chaperone-dependent manner. As a key prediction of a model integrating autocatalytic protein aggregation and a minimal Start network, enforced protein aggregation causes a severe reduction in lifespan, an effect that is greatly alleviated by increased expression of specific chaperones or cyclin Cln3. Overall, our data show that proteostasis breakdown, by compromising chaperone activity and G1-cyclin function, causes an irreversible arrest in G1, configuring a molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence.