Protein Kinase CK2: a major regulator of the G1/S transition in Saccharomyces cerevisiae

Casein kinase 2 (CK2) is a ubiquitous, essential and highly conserved eukaryotic kinase. It phosphorylates more than 300 substrates, but its physiological role and regulation mechanism are still poorly understood (Meggio and Pinna, 2003). CK2 is traditionally considered to be a tetrameric enzyme, composed of two catalytic subunits and two regulatory subunits, which are encoded in yeast by CKA1 and CKA2 genes, CKB1 and CKB2 genes respectively. Deletion of regulatory subunits, or of either catalytic subunit gene alone has few phenotypic effects, but simultaneous disruption of both CKA1 and CKA2 genes is lethal. In Saccharomyces cerevisiae a specialization of the two catalytic subunits (α e α’) was shown: α subunit is involved in cell polarity, while α’ subunit is linked to cell-cycle regulation and was shown to be fundamental both in G1 phase and in mitosis. In fact, at restrictive temperature cka1Δcka2ts mutants arrest cell cycle with a terminal phenotype constituted of 50% of unbudded G1 cells and 50% of cells arrested in metaphase and anaphase (Hanna et al., 1995). Among CK2 substrates, many cell-cycle proteins are known, both in mammalian cells and in yeast (p21, p27, Cdc2, Cdk1, Cdc37) (Meggio and Pinna, 2003). CK2 also phosphorylates two key regulators of the G1/S transition in yeast: Sic1, the Cdk1-Clb5/6 inhibitor, which is phosphorylated by CK2 on Ser201 (Coccetti et al., 2004; Coccetti et al., 2006), and Cdc34, the E2 enzyme required for the ubiquitination of many cell-cycle proteins (among which Sic1), which is phosphorylated by CK2 on Ser207, Ser216 e Ser282 (Pyerin et al., 2005; Barz et al., 2006; Sadowski et al., 2007). My PhD research activity was focused on the relationship between CK2 and these two relevant substrates (Sic1 and Cdc34), in order to understand CK2-mediated regulation of the G1/S transition in yeast. Previous analysis showed an increase of Sic1 level in cka1Δcka2ts strain at 37°C (Coccetti et al., 2006); thus we investigated whether this increase was responsible for the G1 arrest of this strain at restrictive temperature. We showed that the observed increase of Sic1 level was responsible for an inhibition of Cdk1-Clb5 kinasic activity. Moreover, SIC1 deletion, like the shutting-down of its expression, in a cka1Δcka2ts background, bypassed the G1 arrest at 37°C, leading to a single cell-cycle arrest, in which cells showed a post-synthetic DNA content. These data, published in 2007 (Tripodi et al., 2007), explained for the first time the molecular mechanism of the G1 block due to CK2 inactivation: Sic1 accumulation inhibits Cdk1-Clb5 complex, thus preventing the onset of DNA replication. We then worked on Cdc34, the E2 enzyme involved in Sic1 degradation. Literature data and our computational analysis revealed that Cdc34 protein present many consensus sites for CK2 phosphorylation. Mass spectrometry (MS) analysis on recombinant Cdc34 phosphorylated in vitro by CK2 showed phosphorylations on the following sites: S130, S167, S188, S195, S207, S282. In particular, among these, S130 and S167, within the catalytic domain of the protein, are highly conserved among Cdc34 homologues in various organisms, and were identified as phosphorylated sites in vivo in a CK2-dependent manner. Through a thiolester assay, we studied Cdc34 (wild-type and Cdc34S130AS167A) binding to ubiquitin in vitro, and we observed that lack of CK2-mediated phosphorylation on S130 and S167 strongly reduced the ubiquitin-charging ability of Cdc34. Subsequent in vivo analysis allowed us to investigate the physiological role of these phosphorylations. We observed that Cdc34S130AS167A overexpression is not able to complement the thermo-sensitive mutation cdc34-2 (Schwob et al., 1994), and determined a double arrest with both pre-replicative and post-replicative DNA content; the G1 block was characterized by Sic1 accumulation and was bypassed by SIC1 deletion. Yet, Cdc34S130AS167A expression to a level comparable to the endogenous protein led to a uniform G1 arrest at restrictive temperature, like the arrest observed in the control strain cdc34-2ts. Thus, these data, published in 2008 (Coccetti et al., 2008), showed that CK2 phosphorylation of the catalytic domain of Cdc34 was required for the function of the enzyme and for the in vivo ubiquitination of its substrates (among which Sic1). A part from the study of CK2 substrate of G1 phase, we investigated if CK2 was regulated by nutritional conditions, which are important for the modulation of the cell-cycle and especially of the G1/S transition. We used yeast strains expressing TAP-tagged CK2 subunits, and we showed that neither total levels of the four subunits, nor their subcellular localization (which is mainly nuclear both in glucose and in ethanol) were modulated by carbon source. Then we measured CK2 activity, with a traditional assay and with a new assay that we developed using recombinant Sic1 as CK2 substrate; we showed that CK2 activity was clearly lower in ethanol growing cells than in glucose growing ones. We further investigated whether this difference in CK2 activity was due to the different growth rate or to the different carbon metabolism of cells growing in glucose or ethanol. To this purpose, we used bioreactor technology, to separately consider growth rate effects and metabolism effects. This system showed that growth rate was the main factor responsible for the modulation of CK2 activity. We also showed, by using mutant strains bearing a deletion in one of the two genes encoding for the catalytic subunits, that both subunits (α e α’) were regulated by nutritional conditions; moreover, α subunit seems to have a higher activity than α’ subunit. Therefore we provided the first evidence of a regulation of CK2 activity in yeast.