The protein kinase Cdr2, related to Nim1/Cdr1 mitotic inducer, regulates the onset of mitosis in fission yeast

Cdc2-Cyclin B, the protein kinase that catalyzes the onset of mitosis, is subject to multiple forms of regulation. In the fission yeast Schizosaccharomyces pombe and most other species, a key mode of Cdc2-Cyclin B regulation is the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the protein kinases Wee1 and Mik1 and removed by the phosphatase Cdc25. These proteins are also regulated, a notable example being the inhibition of Wee1 by the protein kinase Nim1/Cdr1. The temperature-sensitive mutation cdc25-22 is synthetic lethal with nim1/cdr1 mutations, suggesting that a synthetic lethal genetic screen could be used to identify novel mitotic regulators. Here we describe that such a screen has identified cdr2(+), a gene that has an important role in the mitotic control. Cdr2 is a 775 amino acid protein kinase that is closely related to Nim1 and mitotic control proteins in budding yeast. Deletion of cdr2 causes a G2-M delay that is more severe than that caused by nim1/cdr1 mutations. Genetic studies are consistent with a model in which Cdr2 negatively regulates Wee1. This model is supported by experiments showing that Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro. Thus, Cdr2 is a novel mitotic control protein that appears to regulate Wee1.     

Localization of the 26S proteasome during mitosis and meiosis in fission yeast

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe. Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis. Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope. The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis. Throughout mitosis the proteasome remained predominantly at the nuclear periphery. During meiosis the proteasome was found to undergo dramatic changes in its localization. Throughout the first meiotic division, the signal is more dispersed over the nucleus. During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation. These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis. Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.     

Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast

Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear. We have purified the fission yeast Cdc7 homologue Hsk1 approximately 30,000-fold, to near homogeneity. Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation. Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo. Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation. The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a co-purifying polypeptide, Dfp1. Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity. We have identified Dfp1 as the fission yeast homologue of budding yeast Dbf4. Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast. Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G1/S transition may be mediated by phosphorylation of Cdc19. Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor.     

Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8

During the splicing process, spliceosomal snRNAs undergo numerous conformational rearrangements that appear to be catalyzed by proteins belonging to the DEAD/H-box superfamily of RNA helicases. We have cloned a new RNA helicase gene, designated DBP2 (DEAH-boxprotein), homologous to the Schizosaccaromyces pombe cdc28(+)/prp8(+) gene involved in pre-mRNA splicing and cell cycle progression. The full-length DBP2 contains 3400 nucleotides and codes for a protein of 1041 amino acids with a calculated mol. wt of 119 037 Da. Transfection experiments demonstrated that the GFP-DBP2 gene product, transiently expressed in HeLa cells, was localized in the nucleus. The DBP2 gene was mapped by FISH to the MHC region on human chromosome 6p21.3, a region where many malignant, genetic and autoimmune disease genes are linked. Because the expression of DBP2 gene in S.pombe prp8 mutant cells partially rescued the temperature-sensitive phenotype, we conclude that DBP2 is a functional human homolog of the fission yeast Cdc28/Prp8 protein.     

The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation

The S. cerevisiae SIS1 gene is essential and encodes a heat shock protein with similarity to the bacterial DnaJ protein. At the nonpermissive temperature, temperature-sensitive sis1 strains rapidly accumulate 80S ribosomes and have decreased amounts of polysomes. Certain alterations in 60S ribosomal subunits can suppress the temperature-sensitive phenotype of sis1 strains and prevent the accumulation of 80S ribosomes and the loss of polysomes normally seen under conditions of reduced SIS1 function. Analysis of sucrose gradients for SIS1 protein shows that a large fraction of SIS1 is associated with 40S ribosomal subunits and the smaller polysomes. These and other results indicate that SIS1 is required for the normal initiation of translation. Because DnaJ has been shown to mediate the dissociation of several protein complexes, the requirement of SIS1 in the initiation of translation might be for mediating the dissociation of a specific protein complex of the translation machinery.     

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.     

Multiple orientation-dependent, synergistically interacting, similar domains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe

Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small ( approximately 570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.     

HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system

A new technique for ablation of atrioventricular nodal reentrant tachycardia, using catheter-directed continuous wave Nd-YAG laser light, 1064 nm, via a novel pin-electrode laser catheter, was applied in 10 patients aged 15-63 years (mean 43 years). A total of 22 laser pulses, 1-5 per patient, at 20 or 30 W, of 10-45 s (mean 27 s) were aimed at the postero-inferior aspect of the tricuspid annulus. In all patients the tachycardia was rendered non-inducible at baseline as well as during orciprenaline administration. The amplitudes of the local atrial potentials diminished from 2.0 +/- 0.5 before to 0.4 +/- 0.4 mV after ablation, atrio-His intervals increased from 73 +/- 7 to 157 +/- 36 ms. Anterograde atrioventricular nodal refractory periods (212 +/- 31 vs 238 +/- 31 ms) and Wenckebach rate (174 +/- 8 vs 167 +/- 8 beats.min-1) did not change significantly (P > 0.05). There were no complications or recurrent arrhythmias in a follow-up of 12-35 (mean 27) months. Anatomically guided laser catheter coagulation of the postero-inferior aspect of the tricuspid valve ring is a safe and effective method for the cure of patients with common atrioventricular reentrant tachycardia.     

Sequence and expression of DmMKLP1, a homolog of the human MKLP1 kinesin-like protein from Drosophila melanogaster

We have isolated the Drosophila gene DmMKLP1, which has a high similarity to members of the mitotic kinesin-like subfamily of kinesin proteins. DmMKLP1 has no known close relatives in the Drosophila genome and can therefore be assumed to be the ortholog of human MKLP1 and hamster CHOI kinesin-like proteins. In situ hybridization reveals a homogeneous maternal expression in the early embryo and a terminally restricted expression pattern at blastoderm stage. Later, the expression becomes increasingly restricted to the developing central nervous system, where it remains expressed at least until the end of embryogenesis.     

Negative regulation of interleukin-1beta-activated neutral sphingomyelinase by protein kinase C in rat mesangial cells

Endogenous ceramide is produced by the action of acidic or neutral sphingomyelinases (SMase) in response to stimuli such as proinflammatory cytokines or other inducers of stress. Interleukin-1beta (IL-1beta) is known to stimulate ceramide formation in rat renal mesangial cells; however, the respective subtype of SMase and its regulation have not been investigated. We found that IL-1beta induced an increase in endogenous ceramide levels via the action of a neutral SMase but not an acidic SMase in rat mesangial cells. Cytokine-induced activation of neutral SMase was inhibited by stimulation of protein kinase C (PKC) by the phorbol ester TPA which caused a reduction of ceramide back to control levels. This inhibitory effect of TPA was reversed by the specific PKC-inhibitor Ro-318220. Long-term incubation (24 h) of mesangial cells with TPA, which downregulates PKC-alpha, -delta, and -epsilon isoenzymes, resulted in a recovery of IL-1beta-stimulated neutral SMase activity as well as ceramide formation. These data implicate an important modulatory function of PKC in ceramide production in IL-1beta-activated mesangial cells.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

Regulation of salt tolerance in fission yeast by a protein-phosphatase-Z-like Ser/Thr protein phosphatase

In the yeast Saccharomyces cerevisiae, Na+ efflux is mediated by the Ena1 ATPase, and the expression of the ENA1 gene is regulated by the Ppz1 and Ppz2 Ser/Thr protein phosphatases. On the contrary, in the fission yeast Schizosaccharomyces pombe, effective output of Na+ is attributed to the H+/Na+ antiporter encoded by the sod2 gene. We have isolated a S. pombe gene (pzh1) that encodes a 515-amino-acid protein that is 78% identical, from residue 193 to the COOH terminus, to the PPZ1 and PPZ2 gene products. Bacterially expressed Pzh1p shows enzymatic characteristics virtually identical to those of recombinant Ppz1p. When expressed in high-copy number from the PPZ1 promoter, the pzh1 ORF rescues the caffeine-induced lytic defect and slightly decreases the high salt tolerance of S. cerevisiae ppz1delta mutants. Disruption of pzh1 yields viable S. pombe cells and has virtually no effect on tolerance to caffeine or osmotic stress, but it renders the cells highly tolerant to Na+ and Li+, and hypersensitive to K+. Although lack of pzh1 results in a 2-3-fold increase in sod2 mRNA, the pzh1 mutation significantly increases salt tolerance in the absence of the sod2 gene, suggesting that the phosphatase also regulates a Sod2-independent mechanism. Therefore, the finding of a PPZ-like protein phosphatase involved in the regulation of salt tolerance in fission yeast reveals unexpected aspects of cation homeostasis in this organism.     

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe

BACKGROUND: The small GTP-binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+, from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1. RESULTS: We found that Rho1 is essential for cell viability and cell polarity using gene disruption and by exogenous expression of botulinum C3 ADP-ribosyltransferase. In cells expressing either a constitutively active Rho1 or a dominant-negative Rho1, actin patches were delocalized. Both the cell wall and secondary septum were thick and stratified in cells expressing the constitutively active Rho1, while the cell wall of cells expressing the dominant-negative Rho1 seemed to be loosely organized. Furthermore, inactivation of Rho1 is apparently required for the separation of daughter cells. Cell fractionation studies suggested that Rho1 is predominantly membrane-bound. Moreover, we observed that Rho1 is localized to the cell periphery and to the septum. CONCLUSIONS: Rho1 is involved in actin patch localization, the control of cell polarity, the regulation of septation, and cell wall synthesis.     

Identification of the ure1+ gene encoding urease in fission yeast

We describe a case of subacute cor pulmonale caused by tumor embolism from a gallbladder carcinoma in a 63-year-old woman. The patient was admitted to hospital with increasing dyspnea. Physical examination and echocardiography showed signs of pulmonary hypertension. She died of circulatory failure. At autopsy microscopic studies revealed tumor embolism in the pulmonary vessels and subsequent lesions causing the lethal pulmonary hypertension. This is the first case report of pulmonary hypertension caused by embolism from a gallbladder carcinoma in the literature worldwide.