Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell‐cycle phase‐dependent degradation in yeast

Abstract

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotes and is responsible for the Ser ⁄ Thr phosphorylation of a large number of protein substrates [1][2][3]. The active holoenzyme is most often a heterotetramer composed of two catalytic a subunits, a (encoded by CKA1) and a¢ (encoded by CKA2), and two regulatory b subunits, b and b¢ in Saccharomyces cerevisiae (CKB1 and CKB2). The enzyme occurs in all possible combinations of a and b subunits [4,5]. In yeast, deletion of the gene for one of the two catalytic subunits has little effect, but deletion of both homologous genes results in loss of viability [6]. To date, more than 300 endogenous CK2 substrates are known to be involved in quite diverse processes, e.g. cell proliferation, signal transduction, transcriptional regulation, translation and metabolism [3]. Despite this eminent role in strictly regulated cellular processes and although CK2 is indispensable for cell life, CK2 activity by itself is apparently unregulated [4,5], although some fluctuation in activity in correlation with cell-cycle progression has been seen in cultured mammalian cells [7,8]. The physiological effect of substrate phosphorylation is surprisingly low with almost all targets described to date [9,10]. In S. cerevisiae, it has been shown using temperature-sensitive CKA2 alleles that protein kinase CK2 is required for passage across the G 2 ⁄ M boundary and for cell-cycle progression through the G 1 phase [11]. Using co-immunoprecipitation combined with MS analysis, we identified the a¢ subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Dcka1 or Dcka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A) 6 mutant proteins. Both mutant alleles (S > A) 6 or (S > D) 6 complement in vivo, however, growth of the RIO1 (S > A) 6 mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D) 6 mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G 1 ⁄ S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.Abbreviations Aky2p, adenylate kinase 2; CK2, casein kinase 2; GST, glutathione S-transferase.

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

“…The measured kinase activities were identical (Fig. 4b), confirming an earlier report that recombinant Rio1 lacking a similar C-terminal region is still active as a kinase in vitro 22 . To exclude a possible contribution from casein kinases CK1 and CK2, shown to co-purify with full-length Rio1 (refs 12,22) and with Rio1 lacking its C-terminal 46 residues 9 , the assay was repeated in the presence of CK1 and CK2 inhibitors.…”

“…A previous report indicated that Rio1, overexpressed from the P GAL10 promoter, becomes degraded in S phase, suggesting Rio1 stability is cell cycle regulated 22 . However, our western blot analyses of yeast endogenously expressing Rio1 showed that its protein levels do not change through the cell cycle (Fig.…”

“…8d). Interestingly, Rio1 autophosphorylation 12,22 was threefold (3.1 ± 0.6) higher in the presence of Rpa43, indicating that Rio1 binding to Rpa43 may have stimulated its own phosphorylation, which in turn may have promoted the phosphorylation of Rpa43 (the protein loading controls for the kinase assay are shown in Supplementary Fig. 9g).…”

Integrating Rio1 activities discloses its nutrient-activated network in Saccharomyces cerevisiae

“…To date, only two direct substrates of Rio1 in yeast are known: Rio1 itself (16,68,69) and Rpa43; a conserved subunit of RNA polymerase I (16). To identify protein interactors, we tagged Rio1 N- or C-terminally with either the LexA- or the Gal4 DNA-binding domain.…”

RioK1, a New Interactor of Protein Arginine Methyltransferase 5 (PRMT5), Competes with pICln for Binding and Modulates PRMT5 Complex Composition and Substrate Specificity

“…A corollary of this model is that mechanisms exist to alter RioK1 and/or pICln protein levels or change their affinity for PRMT5 and thereby impart spatial and temporal control over substrate specificity of the PRMT5 complex. Indeed, yeast Rio1p is degraded at the G 1 /S transition of the cell cycle in a casein kinase 2 (CK2)-dependent manner (50). This, together with the fact that human RioK1 is a CK2 substrate in vitro 5 suggests that a similar mechanism may control RioK1 protein levels in human cells.…”