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To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1gene, but not of CLN2, abolished the transient G1phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1gene. Transcription of CLN1is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4gene abolished the transient G1arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4allele mutated in the consensus site for PKA (Swi4S159A) and we found that expression of Swi4S159Aprotein in the GG104-Swi4Δ strain did not restore the transient G1arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1gene expression and G1/S phase transition.

The Saccharomyces cerevisiaeSnf1 protein kinase has been reported to be required for adaptation to glucose limitation and for growth on non-fermentable carbon sources. Here we present novel findings indicating that Snf1, the key regulator of cellular energy, is also involved in yeast cell cycle control. The lack of Snf1 α-catalytic subunit down-regulates the growth rate and CLB5 expression, delaying Sld2 phosphorylation and G1/S transition, in cells grown in 2%, but not in 5% glucose. A non-phosphorylatable Snf1 rescues the slow growth phenotype, whereas a wild type or a phosphomimetic mutant is required to rescue growth rate and the G1/S delay. Using either Snf1 or Swi6 as a bait, a specific interaction of Snf1 with Swi6, the regulatory subunit of MBF, was detected. In conclusion, this report describes a previously unrecognized role for Snf1 in transcriptional modulation of the G1to S transition, differing from the reported AMPK role in controlling the G1/S transition in multicellular eukaryotes.

The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Δcka2-8ts strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34S130AS167A mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2ts yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.