Claiborne V.C. Glover

Casein Kinase II Is Required for Cell Cycle Progression during G1 and G2/M in Saccharomyces cerevisiae

The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% large-budded cells. The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G1. Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G1, at a point which must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle.

ON THE PHYSIOLOGICAL ROLE OF CASEIN KINASE II IN SACCHAROMYCES CEREVISIAE

Casein kinase II (CKII) is a highly conserved serine/threonine protein kinase that is ubiquitous in eukaryotic organisms. This review summarizes available data on CKII of the budding yeast Saccharomyces cerevisiae, with a view toward defining the possible physiological role of the enzyme. Saccharomyces cerevisiae CKII is composed of two catalytic and two regulatory subunits encoded by the CKA1, CKA2, CKB1, and CKB2 genes, respectively. Analysis of null and conditional alleles of these genes identifies a requirement for CKII in at least four biological processes: flocculation (which may reflect an effect on gene expression), cell cycle progression, cell polarity, and ion homeostasis. Consistent with this, isolation of multicopy suppressors of conditional cka mutations has identified three genes that have a known or potential role in either the cell cycle or cell polarity: CDC37, which is required for cell cycle progression in both G1 and G2/M; ZDS1 and 2, which appear to have a function in cell polarity; and SUN2, which encodes a protein of the regulatory component of the 26S protease. The identity and properties of known CKII substrates in S. cerevisiae are also reviewed, and advantage is taken of the complete genomic sequence to predict globally the substrates of CKII in this organism. Although the combined data do not yield a definitive picture of the physiological role of CKII, it is proposed that CKII serves a signal transduction function in sensing and/or communicating information about the ionic status of the cell to the cell cycle machinery.

A Positive Feedback Loop between Protein Kinase CKII and Cdc37 Promotes the Activity of Multiple Protein Kinases

We report here the identification ofCDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13 ts) of the CKA2 gene encoding the α′ catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, onlyCDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylationin vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A andcdc37-S17A mutants at 37 °C, and cdc37-S14Aor cdc37-S14,17A overexpression was incapable of protectingcka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G1 and G2/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.

Cloning and Disruption of CKB1, the Gene Encoding the 38-kDa Subunit of Saccharomyces cerevisiae Casein Kinase II (CKII) DELETION OF CKII REGULATORY SUBUNITS ELICITS A SALT-SENSITIVE PHENOTYPE

Saccharomyces cerevisiae casein kinase II (CKII) contains two distinct catalytic (α and α′) and regulatory (β and β′) subunits. We report here the isolation and disruption of the gene, CKB1, encoding the 38-kDa β subunit. The predicted Ckb1 sequence includes the N-terminal autophosphorylation site, internal acidic domain, and potential metal binding motif (CPX3C-X-CPXC) present in other β subunits but is unique in that it contains two additional autophosphorylation sites as well as a 30-amino-acid acidic insert. CKB1 is located on the left arm of chromosome VII, approximately 33 kilobases from the centromere and does not correspond to any previously characterized genetic locus. Haploid and diploid strains lacking either or both β subunit genes are viable, demonstrating that the regulatory subunit of CKII is dispensable in S. cerevisiae. Such strains exhibit wild type behavior with regard to growth on both fermentable and nonfermentable carbon sources, mating, sporulation, spore germination, and resistance to heat-shock and nitrogen starvation, but are salt-sensitive. Salt sensitivity is specific for NaCl and LiCl and is not observed with KCl or agents which increase osmotic pressure alone. These data suggest a role for CKII in ion homeostasis in S. cerevisiae.

Temperature-sensitive mutations of the CKA1 gene reveal a role for casein kinase II in maintenance of cell polarity in Saccharomyces cerevisiae.

Casein kinase II (CKII) of Saccharomyces cerevisiae contains two distinct catalytic subunits, α and α′, that are encoded by the CKA1 and -2genes, respectively. We have constructed conditional alleles of theCKA1 gene. In contrast to cka1 cka2 ts strains, which exhibit a defect in both G1 and G2/M cell cycle progression,cka1 ts cka2 strains continue to divide for three cell cycles after a shift to restrictive temperature and then arrest as a mixture of budded and unbudded cells with a spherical morphology. Arrested cells exhibit continued growth, a nonpolarized actin cytoskeleton, delocalized chitin deposition, and a significant fraction of multinucleate cell bodies, confirming the presence of a cell polarity defect in cka1 tsstrains. The presence of budded as well as unbudded cells in the arrested population suggests that CKII is required for maintenance rather than establishment of cell polarity, although a role in both processes is also possible. The terminal phenotype ofcka1 ts strains bears a strong resemblance to that of orb5 strains of Schizosaccharomyces pombe, which carry a temperature-sensitive CKII catalytic subunit mutation, but the underlying mechanism appears to be different in the two cases. These results establish a requirement for CKII in cell polarity in S. cerevisiae and provide the first evidence for functional specialization of CKA1 and-2.

A global view of CK2 function and regulation

The wealth of biochemical, molecular, genetic, genomic, and bioinformatic resources available in S. cerevisiae make it an excellent system to explore the global role of CK2 in a model organism. Traditional biochemical and genetic studies have revealed that CK2 is required for cell viability, cell cycle progression, cell polarity, ion homeostasis, and other functions, and have identified a number of potential physiological substrates of the enzyme. Data mining of available bioinformatic resources indicates that (1) there are likely to be hundreds of CK2 targets in this organism, (2) the majority of predicted CK2 substrates are involved in various aspects of global gene expression, (3) CK2 is present in several nuclear protein complexes predicted to have a role in chromatin structure and remodeling, transcription, or RNA metabolism, and (4) CK2 is localized predominantly in the nucleus. These bioinformatic results suggest that the observed phenotypic consequences of CK2 depletion may lie downstream of primary defects in chromatin organization and/or global gene expression. Further progress in defining the physiological role of CK2 will almost certainly require a better understanding of the mechanism of regulation of the enzyme. Beginning with the crystal structure of the human CK2 holoenzyme, we present a molecular model of filamentous CK2 that is consistent with earlier proposals that filamentous CK2 represents an inactive form of the enzyme. The potential role of filamentous CK2 in regulation in vivo is discussed.

The three major types of CRISPR‐Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus

CRISPR‐Cas systems are small RNA‐based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR‐Cas systems including representatives from the three major types of CRISPR‐Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non‐coding RNAs) associated with the respective locus. We find that the Csm Type III‐A and Cse Type I–E crRNAs are specifically processed by Cas6 and Cse3 (Cas6e), respectively, and retain an 8‐nucleotide CRISPR repeat sequence tag 5′ of the invader‐targeting sequence. The Cse Type I–E crRNAs also retain a 21‐nucleotide 3′ repeat tag. The crRNAs from the two Csn Type II‐A systems in Sth consist of a 5′‐truncated targeting sequence and a 3′ tag; however, these are distinct in size between the two. Moreover, the Csn1 (Cas9) protein associated with one Csn locus functions specifically in the production of crRNAs from that locus. Our findings indicate that multiple CRISPR‐Cas systems can function independently in crRNA biogenesis within a given organism – an important consideration in engineering coexisting CRISPR‐Cas pathways.

Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis

CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry.

The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease

Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.

Genes targeted by protein kinase CK2: a genome-wide expression array analysis in yeast.

Protein kinase CK2, a tetramer composed of two catalytically active (CK2α isoforms) and two regulatory (CK2β isoforms) subunits, is suspected to have, among others, a role in gene transcription. To identify the genes targeted by CK2, the transcriptional effect of silencing the CK2 subunit genes in Saccharomyces cerevisiae (CK2α isoform genes: CKA1 and CKA2; CK2β isoform genes: CKB1 and CKB2) was examined using genome-wide expression array analysis (oligonucleotide array chips). Silencing did not influence the overwhelming majority (5801) of the over six thousand open reading frames composing the yeast genome. Cells knocked-out for both CKA1 and CKA2 and plasmid-rescued by Ckal affected specifically at 2-fold discrimination level the transcription of 57 genes, and when rescued by Cka2, the transcription of 118 genes. In CKB1/CKB2 double knock-outs, transcription of 54 genes was specifically altered. Interestingly, aside overlaps between the gene spectra affected by CKA1 and CKA2 silencing, there were overlaps also between those influenced by CK2α and CK2β isoform silencing. The data indicate a distinct role of CK2 in gene transcription control, identify specific functional differences between the two catalytic subunits in gene targeting, and reveal independent effects by the regulatory subunits. (Mol Cell Biochem 227: 59–66, 2001)