| 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 the cell cycle. Due to the conserved regulatory pathways controlling G1 transcription between yeast and mammalian cells, investigation into the regulation by phosphorylation of Whi5 in yeast could provide insight into mammalian G1 cell cycle and Rb regulation.;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 sites that were phosphorylated. Whi5 CDK and non-CDK phosphorylation mutants were functional and able to rescue the small cell size of whi5Delta cells. However, the Whi5 CDK mutant with all 12 putative CDK sites changed to alanine caused a dramatic cell cycle phenotype when overexpressed 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 CDK sites did not wholly determine Whi5 nuclear export, they did impact regulation of cell size. Taken together, these observations begin to dissect the regulatory role of specific phosphorylation sites on Whi5. |
