Whi5 Regulation by Site Specific CDK-Phosphorylation in Saccharomyces cerevisiae

Abstract

The Whi5 transcriptional repressor is a negative regulator of G1 cell cycle progression in Saccharomyces cerevisiae and is functionally equivalent to the Retinoblastoma (Rb) tumor suppressor protein in mammals. In early G1, Whi5 binds to and inhibits SBF (Swi4/Swi6) transcriptional complexes. At Start, Cln:Cdc28 kinases phosphorylate and inactivate Whi5, causing its dissociation from SBF promoters and nuclear export, allowing activation of SBF transcription and entry into late G1. In an analysis of Whi5 phosphorylation, we found that 10 of the 12 putative CDK phosphorylation sites on Whi5 were occupied in vivo in asynchronously growing cells. In addition, we identified 6 non-CDK Whi5 phosphorylation sites. Whi5 CDK and non-CDK phosphorylation mutants were functional and able to rescue the small cell size of whi5Δ cells. However, the Whi5 CDK mutant with all 12 putative CDK sites changed to alanine causes a dramatic cell cycle phenotype when expressed with a Swi6 CDK phosphorylation mutant. Mutational analysis of Whi5 determined that only four C-terminal CDK sites were necessary and sufficient for Whi5 inactivation when Swi6 CDK sites were also mutated. Although these four Whi5 CDK sites do not wholly determine Whi5 nuclear export, they do impact regulation of cell size. Taken together, these observations begin to dissect the regulatory role of specific phosphorylation sites on Whi5.

A Docking Interface in the Cyclin Cln2 Promotes Multi-site Phosphorylation of Substrates and Timely Cell-Cycle Entry

“…Using these conditions, we monitored Whi5 gel mobility (Figure 6A), which reflects CDK phosphorylation [9, 10, 12]. V5-tagged Whi5 was resolved into three species: one in G1 arrested cells, plus two higher forms that appeared after release (Figure 6Ai–ii).…”

Phosphate-Activated Cyclin-Dependent Kinase Stabilizes G1 Cyclin To Trigger Cell Cycle Entry

“…When Cdc28 is associated with Cln3 cyclin, it unfolds the activation of transcriptional G 1 machinery, chiefly through two mechanisms: phosphorylation of the Whi5 repressor (and presumably of Swi6) at multiple residues (10)(11)(12) and control of the Stb1 dual protein and the histone deacetylases (13). In addition to Cln3, other elements involved in G 1 transcription firing have recently been demonstrated: for example, a positive-feedback mechanism involving Cln1 and Cln2 could reinforce SBF/MBF activation (14).…”

Protein Kinase CK2 Holoenzyme Promotes Start-Specific Transcription in Saccharomyces cerevisiae

“…Alternatively, the CK2 holoenzyme may be involved in the phosphorylation of different proteins within the transcriptional complex to exert its regulatory activity. This is not surprising considering that also the Whi5-12Ala mutant (with all 12 Cdk1 sites changed to alanine) shows no alteration in cell cycle progression unless it is co-overexpressed with the Swi6-Cdk1 (Swi6-4Ala) phosphorylation mutant (13). Therefore, the CK2 holoenzyme may stimulate gene expression by phosphorylating different key proteins and then acting at several levels of the transcriptional process.…”

“…The specific and coherent expression of G 1 -specific genes is achieved by the interaction of SBF and MBF complexes with many different regulators, such as chromatin remodelling factors (Rpd3, Hos3, and the FACT complex), coactivators, and repressors (Stb1 and Whi5) (6)(7)(8)(9)(10)(11)(12)(13). The combined actions of all these proteins allow the recruitment of the mediator complex, the RNA polymerase II complex, and several general transcription factors (GTFs), which ultimately leads to transcription initiation.…”