Whi5 Regulation by Site Specific CDK-Phosphorylation in Saccharomyces cerevisiae

Mikhail V Blagosklonny,Michelle V Wagner,Huilin Zhou,Rob A M De Bruin,Curt Wittenberg,Marcus B Smolka,Steven F Dowdy

doi:10.1371/journal.pone.0004300

Mikhail V Blagosklonny, Michelle V Wagner + Show 5 more

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https://doi.org/10.1371/journal.pone.0004300

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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.

Highlights

Cell proliferation is a tightly regulated process where extracellular signals and intracellular checkpoints are integrated to control cell growth and division
Whi5 contains 12 putative cyclin dependent kinase (CDK) phosphorylation sites typified by S/T-P(-X-B) and phosphorylation by Cln:Cdk activity has been shown to be important in Whi5 inactivation [9,10]. de Bruin et al had previously determined that CDK sites 2, 4, 5, 10 and 12 were
Based on a preliminary survey of phosphorylation site mutants, we initially focused our attention on the C-terminal CDK phosphorylation sites

Summary

Introduction

Cell proliferation is a tightly regulated process where extracellular signals and intracellular checkpoints are integrated to control cell growth and division. Growth factor signaling determines passage through the Restriction Point that is presumed to be partially responsive to cell size and mass. The molecular events determining commitment to cell division in both yeast and mammalian cells involve regulated transcription of groups of genes required for cell cycle progression [5,6]. SBF controls transcription of cell cycle regulatory genes, while genes involved in DNA synthesis and repair are MBF targets [7]. Loss of both Swi and Mbp results in a permanent G1 arrest and cell lethality, demonstrating the essential function of these transcription factor complexes [8]

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