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

Using co-immunoprecipitation combined with MS analysis, we identified the alpha' 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 Deltacka1 or Deltacka2 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.     

Crystal structure of A. fulgidus Rio2 defines a new family of serine protein kinases

The RIO family of atypical serine/threonine kinases contains two subfamilies, Rio1 and Rio2, highly conserved from archaea to man. Both RIO proteins from Saccharomyces cerevisiae catalyze serine phosphorylation in vitro, and the presence of conserved catalytic residues is required for cell viability. The activity of Rio2 is necessary for rRNA cleavage in 40S ribosomal subunit maturation. We solved the X-ray crystal structure of Archaeoglobus fulgidus Rio2, with and without bound nucleotides, at 2.0 A resolution. The C-terminal RIO domain is indeed structurally homologous to protein kinases, although it differs from known serine kinases in ATP binding and lacks the regions important for substrate binding. Unexpectedly, the N-terminal Rio2-specific domain contains a winged helix fold, seen primarily in DNA-binding proteins. These discoveries have implications in determining the target and function of RIO proteins and define a distinct new family of protein kinases.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31°?C. Cells are temperature sensitive at 35.5°?C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19°?C to 25°?C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.     

Dominant Rio1 kinase/ATPase catalytic mutant induces trapping of late pre-40S biogenesis factors in 80S-like ribosomes

During eukaryotic ribosome biogenesis, members of the conserved atypical serine/threonine protein kinase family, the RIO kinases (Rio1, Rio2 and Rio3) function in small ribosomal subunit biogenesis. Structural analysis of Rio2 indicated a role as a conformation-sensing ATPase rather than a kinase to regulate its dynamic association with the pre-40S subunit. However, it remained elusive at which step and by which mechanism the other RIO kinase members act. Here, we have determined the crystal structure of the human Rio1–ATP–Mg²⁺ complex carrying a phosphoaspartate in the active site indicative of ATPase activity. Structure-based mutations in yeast showed that Rio1''s catalytic activity regulates its pre-40S association. Furthermore, we provide evidence that Rio1 associates with a very late pre-40S via its conserved C-terminal domain. Moreover, a rio1 dominant-negative mutant defective in ATP hydrolysis induced trapping of late biogenesis factors in pre-ribosomal particles, which turned out not to be pre-40S but 80S-like ribosomes. Thus, the RIO kinase fold generates a versatile ATPase enzyme, which in the case of Rio1 is activated following the Rio2 step to regulate one of the final 40S maturation events, at which time the 60S subunit is recruited for final quality control check.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31° C. Cells are temperature sensitive at 35.5° C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19° C to 25° C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest. Received: 14 October 1998 / Accepted: 15 March 1999     

Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.

The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis.     

Mutational analysis of the fission yeast p34cdc2 protein kinase gene

Summary The p34^cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34^cdc2 (each ca. 60 % identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34^cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34^cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with trypthophan renders the resulting mutant protein p80^cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34^cdc2 function. Five acidic amino acids have also been mutated within p34^cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34^cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34^cdc2 enzyme.     

Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint

The morphogenesis checkpoint in budding yeast delays cell cycle progression in G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G(2) arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28(Y19F) allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28(E12K) allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28(Y19F) allele suggested that Swe1p is still able to inhibit CDC28(Y19F) in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms.     

4alpha-Phorbol negates the inhibitory effects of phorbol-12-myristate-13-acetate on human cilia and alters the phosphorylation of PKC

RIO1 and Rio-related proteins display little similarity of primary sequence with conventional protein kinases. Based on secondary structure alignments, we show that it contains the domain structure (subdomains I-XI) and conserved secondary structure elements found in conventional protein kinases. We show that recombinant wild-type Rio1p isolated from Escherichia coli displays kinase activity which depends on autophosphorylation and magnesium or manganese as ATP-activating ions. An initial biochemical characterization of Rio1p is presented.     

A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast

Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase. This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit controlled by the anaphase-promoting complex (APC). However, an abnormal spindle and/or unattached kinetochores activates a conserved spindle checkpoint that blocks APC function. This leads to high mitotic kinase activity and prevents mitotic exit. DBF2 belongs to a group of budding yeast cell cycle genes that when mutated prevent cyclin degradation and block exit from mitosis. DBF2 encodes a protein kinase which is cell cycle regulated, peaking in metaphase-anaphase B/telophase, but its function remains unknown. Here, we show the Dbf2p kinase activity to be a target of the spindle checkpoint. It is controlled specifically by Bub2p, one of the checkpoint components that is conserved in fission yeast and higher eukaroytic cells. Significantly, in budding yeast, Bub2p shows few genetic or biochemical interactions with other members of the spindle checkpoint. Our data now point to the protein kinase Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p blocks Dbf2p function.     

Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein

Numerous non-ribosomal trans-acting factors involved in pre-ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation. Here we demonstrate that Rio1p/Rrp10p is such a factor. Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man. We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation. We show that RIO1 encodes a cytoplasmic non-ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre-rRNA accumulation. In situ hybridization reveals that, in Rio1p depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre-rRNA at site D, producing mature 18S rRNA. Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10). Rio1p/Rrp10p is the first non-ribosomal factor characterized specifically required for 20S pre-rRNA processing.     

A Novel Member of the Cyclin-Dependent Kinase Family in Paramecium tetraurelia

Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.     

Interaction of the pim1/spi1 mitotic checkpoint with a protein phosphatase.

Loss of p58pim1, a homolog of human RCC1, results in uncoupling of mitosis from the completion of DNA replication in fission yeast. An extragenic suppressor of a mutant allele of pim1, esp1, has been isolated and characterized. esp1 encodes a predicted product of 305 amino acid residues, which shares 71% identity with budding yeast SIT4, a type2A related protein phosphatase. p58pim1 binds p25spi1, a 25-kd ras-related GTPase previously isolated as a high dosage suppressor of pim1. The complex dissociates in the presence of guanine nucleotides and Mg2+. The mutant p58pim1 is defective in its ability to bind p25spi1, suggesting that the physical interaction is essential for the maintenance of the interdependency of cell cycle event. In the esp1 pim1 double mutant, the mutant p58pim1 protein is still defective in its ability to bind to p25spi1. However, pmi1 induced premature mitosis is completely suppressed, suggesting that esp1 may act downstream of the p58pim1/p25spi1 physical interaction but upstream of the activation of the M-phase specific histone H1 kinase.     

Structure and activity of the atypical serine kinase Rio1

Rio1 is the founding member of the RIO family of atypical serine kinases that are universally present in all organisms from archaea to mammals. Activity of Rio1 was shown to be absolutely essential in Saccharomyces cerevisiae for the processing of 18S ribosomal RNA, as well as for proper cell cycle progression and chromosome maintenance. We determined high-resolution crystal structures of Archaeoglobus fulgidus Rio1 in the presence and absence of bound nucleotides. Crystallization of Rio1 in the presence of ATP or ADP and manganese ions demonstrated major conformational changes in the active site, compared with the uncomplexed protein. Comparisons of the structure of Rio1 with the previously determined structure of the Rio2 kinase defined the minimal RIO domain and the distinct features of the RIO subfamilies. We report here that Ser108 represents the sole autophosphorylation site of A. fulgidus Rio1 and have therefore established its putative peptide substrate. In addition, we show that a mutant enzyme that cannot be autophosphorylated can still phosphorylate an inactive form of Rio1, as well as a number of typical kinase substrates.     

Functional implication of human serine/threonine kinase, hAIK, in cell cycle progression

Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines. The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm). During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes. In addition, overexpression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive. Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures.     

A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.     

Premature chromatin condensation upon accumulation of NIMA.

The NIMA protein kinase of Aspergillus nidulans is required for the G2/M transition of the cell cycle. Mutants lacking NIMA arrest without morphological characteristics of mitosis, but they do contain an activated p37nimX kinase (the Aspergillus homologue of p34cdc2). To gain a better understanding of NIMA function we have investigated the effects of expressing various NIMA constructs in Aspergillus, fission yeast and human cells. Our experiments have shown that the instability of the NIMA protein requires sequences in the non-catalytic C-terminus of the protein. Removal of this domain results in a stable protein that, once accumulated, promotes a lethal premature condensation of chromatin without any other aspects of mitosis. Similar effects were also observed in fission yeast and human cells accumulating Aspergillus NIMA. This phenotype is independent of cell cycle progression and does not require p34cdc2 kinase activity. As gain of NIMA function by accumulation results in premature chromatin condensation, and loss of NIMA function results in an inability to enter mitosis, we propose that NIMA functions in G2 to promote the condensation of chromatin normally associated with entry into mitosis.     

Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans

We show that in Aspergillus nidulans, p34cdc2 tyrosine dephosphorylation accompanies activation of p34cdc2 as an H1 kinase at mitosis. However, the nimA5 mutation arrests cells in G2 with p34cdc2 tyrosine dephosphorylated and fully active as an H1 kinase. Activation of NIMA is therefore not required for p34cdc2 activation. Furthermore, mutation of nimT, which encodes a protein with 50% similarity to fission yeast cdc25, causes a G2 arrest and prevents tyrosine dephosphorylation of p34cdc2 but does not prevent full activation of the NIMA protein kinase. Mitotic activation of p34cdc2 by tyrosine dephosphorylation is therefore not required for activation of NIMA. These data suggest that activation of either the p34cdc2 protein kinase or the NIMA protein kinase alone is not sufficient to initiate mitosis. Parallel activation of both cell cycle-regulated protein kinases is required to trigger mitosis.     

Alk1 and Alk2 are Two New Cell Cycle-Regulated Haspin-Like Proteins in Budding Yeast

Haspin is a protein kinase identified in mouse and human cells, and genes coding forhaspin-like proteins are present in virtually all eukaryotic genomes sequenced so far. Twohaspin homologues, called Alk1 and Alk2, are present in the yeast Saccharomycescerevisiae. Both Alk1 and Alk2 exhibit a weak auto-kinase activity in vitro, arephosphoproteins in vivo and are hyperphosphorylated in response to DNA damage. Theamount and modification of the two proteins is greatly regulated during the cell cycle. Infact, Alk1 and Alk2 levels peak in mitosis and late-S/G2, respectively, and phosphorylationof both proteins is maximal in mitosis. Control of protein stability plays a major role inAlk2 regulation. The half-life of Alk2 is particularly short in G1; mutagenesis and geneticanalysis indicate that its degradation is controlled by the APC pathway. Overexpression ofALK2, but not of ALK1, causes a mitotic arrest, which is correlated to the kinase activity ofthe protein. This finding, together with its cell cycle regulation, suggests a role for Alk2 inthe control of mitosis.     

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.