Secondary literature sources for TRP_N
The following references were automatically generated.
- Schonbrun M, Laor D, Lopez-Maury L, Bahler J, Kupiec M, Weisman R
- TOR complex 2 controls gene silencing, telomere length maintenance, and survival under DNA-damaging conditions.
- Mol Cell Biol. 2009; 29: 4584-94
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The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Deltator1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.
- Aydar E, Palmer CP
- Polycystic kidney disease channel and synaptotagmin homologues play roles in schizosaccharomyces pombe cell wall synthesis/repair and membrane protein trafficking.
- J Membr Biol. 2009; 229: 141-52
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Eukaryotic cells can sense a wide variety of environmental stresses, including changes in temperature, pH, osmolarity and nutrient availability. They respond to these changes through a variety of signal-transduction mechanisms, including activation of Ca(2+)-dependent signaling pathways. This research has discovered important implications in the function(s) of polycystic kidney disease (PKD) channels and the mechanisms through which they act in the control of cell growth and cell polarity in Schizosaccharomyces pombe by ion channel-mediated Ca(2+) signaling. Pkd2 was expressed maximally during the exponential growth phase. At the cell surface pkd2 was localized at the cell tip during the G(2) phase of the cell cycle, although following cell wall damage, the cell surface-expressed protein relocalized to the whole plasma membrane. Pkd2 depletion affected Golgi trafficking, resulting in a buildup of vesicles at the cell poles, and strongly affected plasma membrane protein delivery. Surface-localized pkd2 was present in the plasma membrane for a very short time and was rapidly internalized. Internalization was dependent on Ca(2+), enhanced by amphipaths and inhibited by gadolinium. The pkd2 protein was in a complex with a yeast synaptotagmin homologue and myosin V. Depletion of pkd2 severely affected the localization of glucan synthase. A role for pkd2 in a cell polarity and cell wall synthesis signaling complex with a synaptotagmin homologue, myosin V and glucan synthase is proposed.
- Venkatachalam K, Montell C
- TRP channels.
- Annu Rev Biochem. 2007; 76: 387-417
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The TRP (Transient Receptor Potential) superfamily of cation channels is remarkable in that it displays greater diversity in activation mechanisms and selectivities than any other group of ion channels. The domain organizations of some TRP proteins are also unusual, as they consist of linked channel and enzyme domains. A unifying theme in this group is that TRP proteins play critical roles in sensory physiology, which include contributions to vision, taste, olfaction, hearing, touch, and thermo- and osmosensation. In addition, TRP channels enable individual cells to sense changes in their local environment. Many TRP channels are activated by a variety of different stimuli and function as signal integrators. The TRP superfamily is divided into seven subfamilies: the five group 1 TRPs (TRPC, TRPV, TRPM, TRPN, and TRPA) and two group 2 subfamilies (TRPP and TRPML). TRP channels are important for human health as mutations in at least four TRP channels underlie disease.
- Qamar S, Vadivelu M, Sandford R
- TRP channels and kidney disease: lessons from polycystic kidney disease.
- Biochem Soc Trans. 2007; 35: 124-8
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Important insights in to the function of members of the TRP (transient receptor potential) channel superfamily have been gained from the identification of disease-related mutations. In particular the identification of mutations in the PKD2 gene in autosomal dominant polycystic kidney disease has revealed a link between TRP channel function, mechanosensation and the role of the primary cilium in renal cyst formation. The PKD2 gene encodes TRPP2 (transient receptor potential polycystin 2) that has significant homology to voltage-activated calcium and sodium TRP channels. It interacts with polycystin-1 to form a large membrane-associated complex that is localized to the renal primary cilium. Functional characterization of this polycystin complex reveals that it can respond to mechanical stimuli such as flow, resulting in influx of extracellular calcium and release of calcium from intracellular stores. TRPP2 is expressed in the endoplasmic reticulum/sarcoplasmic reticulum where it also regulates intracellular calcium signalling. Therefore TRPP2 modulates many cellular processes via intracellular calcium-dependent signalling pathways.
- Huang K, Diener DR, Mitchell A, Pazour GJ, Witman GB, Rosenbaum JL
- Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella.
- J Cell Biol. 2007; 179: 501-14
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To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2-GFP, about 10% of flagellar CrPKD2-GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2-GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.
- Champion A et al.
- AtSGP1, AtSGP2 and MAP4K alpha are nucleolar plant proteins that can complement fission yeast mutants lacking a functional SIN pathway.
- J Cell Sci. 2004; 117: 4265-75
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In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is signalled via the septation initiation network (SIN) involving several protein kinases and a GTPase. Arabidopsis thaliana and Brassica napus proteins homologous to fission yeast spg1p (AtSGP1, AtSGP2), cdc7p (AtMAP3K epsilon 1, AtMAP3K epsilon 2, BnMAP3K epsilon 1) and sid1p (AtMAP4K alpha 1, AtMAP4K alpha 2, BnMAP4K alpha 2) exhibit a significant similarity. The plant proteins AtSGP1/2 and BnMAP4K alpha 2 are able to complement the S. pombe mutant proteins spg1-B8 and sid1-239, respectively and to induce mutisepta when overexpressed in wild-type yeast. Yeast two-hybrid assays demonstrated interactions both between plant proteins and between plant and yeast proteins of the SIN pathway. However, the primary structure of the proteins as well as the partial complementation of yeast mutants indicates that plant homologous proteins and their yeast counterparts have diverged during evolution. Real-time RT-PCR studies demonstrated plant SIN-related gene expression in all organs tested and a co-expression pattern during the cell cycle, with a higher accumulation at G(2)-M. During interphase, the plant SIN-related proteins were found to co-localise predominantly in the nucleolus of the plant cells, as shown by fusions to green fluorescent protein. These data suggest the existence of a plant SIN-related pathway.
- Tajadura V, Garcia B, Garcia I, Garcia P, Sanchez Y
- Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall beta-glucan biosynthesis through the GTPase Rho1p.
- J Cell Sci. 2004; 117: 6163-74
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Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell-wall synthesis. Here, we describe the cloning and characterization of rgf3+, a member of the Rho family of guanine nucleotide exchange factors (Rho GEFs). The rgf3+ gene was cloned by complementation of a mutant (ehs2-1) hypersensitive to drugs that interfere with cell-wall biosynthesis. The rgf3+ gene was found to be essential for cell viability and depletion of Rgf3p afforded phenotypes similar to those obtained following depletion of Rho1p. However, the cell death caused by Rgf3p depletion could be rescued by the presence of 1.2 M sorbitol, whereas depletion of Rho1 was lethal under the same conditions. We show that Rgf3p is a specific Rho1-GEF. The hypersensitivity to drugs affecting the cell wall of the ehs2-1 mutant was suppressed by overexpression of rho1+ but not by any of the other GTPases of the Rho family. Rgf3p interacted with the GDP-bound form of Rho1p and promoted the GDP-GTP exchange. In addition, we show that overexpression of Rgf3p produces multiseptated cells and increases beta-1,3-glucan synthase activity and the amount of cell wall beta-1,3-glucan. Rgf3p localized to the septum and the mRNA level was regulated in a cell-cycle-dependent manner peaking during septation. Our results suggest that Rgf3p acts as a positive activator of Rho1p, probably activating the Rho functions that coordinate cell-wall biosynthesis to maintain cell integrity during septation.
- Stokes NR et al.
- A role for mechanosensitive channels in survival of stationary phase: regulation of channel expression by RpoS.
- Proc Natl Acad Sci U S A. 2003; 100: 15959-64
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The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by Escherichia coli cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of mscL- and mscS-LacZ fusions in an rpoS mutant strain suggested that the RNA polymerase holoenzyme containing sigmaS is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of sigmaS is direct, the MscS and MscL promoters both use RNA polymerase containing sigmaS in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of sigmaS, show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.
- Hariharan IK, Haber DA
- Yeast, flies, worms, and fish in the study of human disease.
- N Engl J Med. 2003; 348: 2457-63
- Clapham DE
- TRP channels as cellular sensors.
- Nature. 2003; 426: 517-24
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TRP channels are the vanguard of our sensory systems, responding to temperature, touch, pain, osmolarity, pheromones, taste and other stimuli. But their role is much broader than classical sensory transduction. They are an ancient sensory apparatus for the cell, not just the multicellular organism, and they have been adapted to respond to all manner of stimuli, from both within and outside the cell.
- Walker SA, Lockyer PJ, Cullen PJ
- The Ras binary switch: an ideal processor for decoding complex Ca2+ signals?
- Biochem Soc Trans. 2003; 31: 966-9
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Activation of cell-surface receptors often leads to changes in intracellular calcium concentration ([Ca(2+)](i)). Receptor-generated calcium transients are often seen as repetitive spikes of elevated intracellular calcium concentration ([Ca(2+)](i)), whose frequency varies according to the amplitude of the receptor stimuli. This suggests a requirement for molecular decoders, capable of interpreting such complex calcium signals into the correct physiological response. Ras proteins are binary molecular switches controlling a plethora of cellular responses. Whether Ras is in its active GTP-bound, or inactive GDP-bound, form is determined by the activity of guanine nucleotide exchange factors (GEFs) and GTPase-activating protein (GAPs). Calcium-regulated GEFs and GAPs have been identified, some with an exquisite sensitivity to [Ca(2+)](i), implicating a potential role of complex calcium signals in regulating Ras.
- Garcia-Salcedo JA, Nolan DP, Gijon P, Gomez-Rodriguez J, Pays E
- A protein kinase specifically associated with proliferative forms of Trypanosoma brucei is functionally related to a yeast kinase involved in the co-ordination of cell shape and division.
- Mol Microbiol. 2002; 45: 307-19
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The life cycle of African trypanosomes is characterized by the alternation of proliferative and quiescent stages but the molecular details of this process remain unknown. Here, we describe a new cytoplasmic protein kinase from Trypanosoma brucei, termed TBPK50, that belongs to a family of protein kinases involved in the regulation of the cell cycle, cell shape and proliferation. TBPK50 is expressed only in proliferative forms but is totally absent in quiescent cells despite the fact that the gene is constitutively transcribed at the same level throughout the life cycle. It is probable that TBPK50 has very specific substrate requirements as it was unable to transphosphorylate a range of classical phosphoacceptor substrates in vitro, although an autophosphorylation activity was readily detectable in the same assays. Complementation studies using a fission yeast mutant demonstrated that TBPK50 is a functional homologue of Orb6, a protein kinase involved in the regulation of cellular morphology and cell cycle progression in yeast. These results link the expression of TBPK50 and the growth status of trypanosomes and support the view that this protein kinase is likely to be involved in the control of life cycle progression and cell division of these parasites.
- Yada T et al.
- Its8, a fission yeast homolog of Mcd4 and Pig-n, is involved in GPI anchor synthesis and shares an essential function with calcineurin in cytokinesis.
- J Biol Chem. 2001; 276: 13579-86
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In fission yeast, calcineurin is required for cytokinesis and ion homeostasis; however, most of its physiological roles remain obscure. To identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature and isolated the mutant its8-1. its8(+) encodes a homolog of the budding yeast MCD4 and human Pig-n that are involved in glycosylphosphatidylinositol (GPI) anchor synthesis. Consistently, reduced inositol labeling of proteins suggested impaired GPI anchor synthesis in its8-1 mutants. The temperature upshift induced a further decrease in inositol labeling and caused dramatic increases in the frequency of septation in its8-1 mutants. BE49385A, an inhibitor of MCD4 and Pig-n, also increased the septation index of the wild-type cell. Osmotic stabilization suppressed these morphological defects, indicating that cell wall weakness caused by impaired GPI anchor synthesis resulted in abnormal cytokinesis. Furthermore, calcineurin-deleted cells exhibited hypersensitivity to BE49385A, and FK506 exacerbated the cytokinesis defects of the its8-1 mutant. Thus, calcineurin and Its8 may share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity.
- Bok JW et al.
- Structure and function analysis of the calcium-related gene spray in Neurospora crassa.
- Fungal Genet Biol. 2001; 32: 145-58
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The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca(2+) via calcineurin.
- Speranca MA et al.
- Primary structure of the Plasmodium vivax crk2 gene and interference of the yeast cell cycle upon its conditional expression.
- Exp Parasitol. 2001; 97: 119-28
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The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle. To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2). The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes. We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies. Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S. pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect. However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature. Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery.
- McLeod M, Shor B, Caporaso A, Wang W, Chen H, Hu L
- Cpc2, a fission yeast homologue of mammalian RACK1 protein, interacts with Ran1 (Pat1) kinase To regulate cell cycle progression and meiotic development.
- Mol Cell Biol. 2000; 20: 4016-27
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The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis. Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis. The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1. Here we report the identification of one of these, Cpc2. Cpc2 encodes a homologue of RACK1, a WD protein with homology to the beta subunit of heterotrimeric G proteins. RACK1 is a highly conserved protein, although its function remains undefined. In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C. Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed. cpc2Delta cells fail to accumulate in G(1) when starved of nitrogen. This leads to defects in conjugation and meiosis. Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro. Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2Delta cells. Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase. All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues.
- Carnero E, Ribas JC, Garcia B, Duran A, Sanchez Y
- Schizosaccharomyces pombe ehs1p is involved in maintaining cell wall integrity and in calcium uptake.
- Mol Gen Genet. 2000; 264: 173-83
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The Schizosaccharomyces pombe mutant ehs1-1 mutant was isolated on the basis of its hypersensitivity to Echinocandin and Calcofluor White, which inhibit cell wall synthesis. The mutant shows a thermosensitive growth phenotype that is suppressed in the presence of an osmotic stabiliser. The mutant also showed other cell wall-associated phenotypes, such as enhanced sensitivity to enzymatic cell wall degradation and an imbalance in polysaccharide synthesis. The ehs1 + gene encodes a predicted integral membrane protein that is 30% identical to Saccharomyces cerevisiae Mid1p, a protein that has been proposed to form part of a calcium channel. As expected for such a function, we found that ehs1+ is involved in intracellular Ca2+ accumulation. High external Ca2+ concentrations suppressed all phenotypes associated with the ehs1 null mutation, suggesting that the cell integrity defects of ehs1 mutants result from inadequate levels of calcium in the cell. We observed a genetic relationship between ehs1+ and the protein kinase C homologue pck2+. pck2+ suppressed all phenotypes of ehs1-1 mutant cells. Overproduction of pck2p is deleterious to wild-type cells, increasing 1,3-beta-D-glucan synthase activity and promoting accumulation of extremely high levels of Ca2+. The lethality associated with pck2p, the increase in 1,3-beta-D-glucan synthase production and the strong Ca2+ accumulation are all dependent on the presence of ehs1p. Our results suggest that in fission yeast ehs1p forms part of a calcium channel that is involved in the cell wall integrity pathway that includes the kinase pck2p.
- Liu J, Kipreos ET
- Evolution of cyclin-dependent kinases (CDKs) and CDK-activating kinases (CAKs): differential conservation of CAKs in yeast and metazoa.
- Mol Biol Evol. 2000; 17: 1061-74
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Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription. CDK activation depends on phosphorylation by a CDK-activating kinase (CAK). Different CAKs have been identified in budding yeast, fission yeast, and metazoans. All known CAKs belong to the extended CDK family. The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, have diverged considerably from other CDKs. Cell cycle regulatory components have been largely conserved in eukaryotes; however, orthologs of neither CAK1 nor csk1 have been identified in other species to date. To determine the evolutionary relationships of yeast and metazoan CAKs, we performed a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, humans, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans. We observed that there were 10 clades for CDK-related genes, of which seven appeared ancestral, containing both yeast and metazoan genes. The four clades that contain CDKs that regulate transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II generally have only a single orthologous gene in each species of yeast and metazoans. In contrast, the ancestral cell cycle CDK (analogous to budding yeast CDC28) gave rise to a number of genes in metazoans, as did the ancestor of budding yeast PHO85. One ancestral clade is unique in that there are fission yeast and metazoan members, but there is no budding yeast ortholog, suggesting that it was lost subsequent to evolutionary divergence. Interestingly, CAK1 and csk1 branch together with high bootstrap support values. We used both the relative apparent synapomorphy analysis (RASA) method in combination with the S-F method of sampling reduced character sets and gamma-corrected distance methods to confirm that the CAK1/csk1 association was not an artifact of long-branch attraction. This result suggests that CAK1 and csk1 are orthologs and that a central aspect of CAK regulation has been conserved in budding and fission yeast. Although there are metazoan CDK-family members for which we could not define ancestral lineage, our analysis failed to identify metazoan CAK1/csk1 orthologs, suggesting that if the CAK1/csk1 gene existed in the metazoan ancestor, it has not been conserved.
- Miller C
- Ion channel surprises: prokaryotes do it again!
- Neuron. 2000; 25: 7-9
- Sayers LG et al.
- Rho-dependence of Schizosaccharomyces pombe Pck2.
- Genes Cells. 2000; 5: 17-27
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BACKGROUND: In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C-related PRKs, is responsible for an interaction with Rho-GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho-dependent protein kinase C-related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2. RESULTS: Rho1 is shown to interact with the conserved N-terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co-expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2-Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located. CONCLUSIONS: The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho-GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.
- Aoyama K, Mitsubayashi Y, Aiba H, Mizuno T
- Spy1, a histidine-containing phosphotransfer signaling protein, regulates the fission yeast cell cycle through the Mcs4 response regulator.
- J Bacteriol. 2000; 182: 4868-74
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Common histidine-to-aspartate (His-to-Asp) phosphorelay signaling systems involve three types of signaling components: a sensor His kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) protein. In the fission yeast Schizosaccharomyces pombe, two response regulators, Mcs4 and Prr1, have been identified recently, and it was shown that they are involved in the signal transduction implicated in stress responses. Furthermore, Mcs4 appears to be involved in mitotic cell-cycle control. However, neither the HPt phosphotransmitter nor His kinase has been characterized in S. pombe. In this study, we identified a gene encoding an HPt phosphotransmitter, named Spy1 (S. pombe YPD1-like protein). The spy1(+) gene showed an ability to complement a mutational lesion of the Saccharomyces cerevisiae YPD1 gene, which is involved in an osmosensing signal transduction. The result from yeast two-hybrid analysis indicated that Spy1 interacts with Mcs4. To gain insight into the function of Spy1, a series of genetic analyses were conducted. The results provided evidence that Spy1, together with Mcs4, plays a role in regulation of the G(2)/M cell cycle progression. Spy1-deficient cells appear to be precocious in the entry to M phase. In the proposed model, Spy1 modulates Mcs4 in a negative manner, presumably through a direct His-to-Asp phosphorelay, operating upstream of the Sty1 mitogen-activated protein kinase cascade.
- Kiely J, Haase SB, Russell P, Leatherwood J
- Functions of fission yeast orp2 in DNA replication and checkpoint control.
- Genetics. 2000; 154: 599-607
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orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.
- Inoue SB, Qadota H, Arisawa M, Watanabe T, Ohya Y
- Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase.
- J Biol Chem. 1999; 274: 38119-24
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One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1, 3-beta-glucan synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-beta-glucan synthase. The 1,3-beta-glucan synthase activity of the cal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1, 3-beta-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. Glutathione S-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-beta-glucan synthase of the rho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-beta-glucan synthase in vitro. Increased cytoplasmic levels of divalent cations such as Ca(2+) restored both Rho1p modification and the 1,3-beta-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.
- Khasanov FK, Savchenko GV, Bashkirova EV, Korolev VG, Heyer WD, Bashkirov VI
- A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.
- Genetics. 1999; 152: 1557-72
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A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.
- Niederberger C, Schweingruber ME
- A Schizosaccharomyces pombe gene, ksg1, that shows structural homology to the human phosphoinositide-dependent protein kinase PDK1, is essential for growth, mating and sporulation.
- Mol Gen Genet. 1999; 261: 177-83
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Fission yeast (Schizosaccharomyces pombe) requires inositol for growth, mating and sporulation. To define putative genes that are involved in the processing and transduction of the inositol signal, mutants that are temperature sensitive for growth and sporulation were selected on a medium containing non-limiting amounts of inositol. Two such mutants (ksg1-208 and ksg1-358) were analyzed, which are impaired in mating and sporulation at 30 degrees C and undergo growth arrest in the G2 phase of the cell cycle at 35 degrees C. The ksg1 gene was isolated by functional complementation. It maps on the left arm of chromosome II and encodes a putative 592-amino acid protein which exhibits good structural homology to a human 3-phosphoinositide-dependent protein kinase (PDK1) and its rat and Drosophila homologues. The two mutants have the same substitution at amino acid position 159: a glycine residue is replaced by glutamic acid. Deletion of the gene is lethal for haploid cells. We propose that ksg1 is involved in one or several phosphoinositide signalling processes that are responsible for control of the life cycle.
- Barr MM, Sternberg PW
- A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans.
- Nature. 1999; 401: 386-9
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The stereotyped mating behaviour of the Caenorhabditis elegans male is made up of several substeps: response, backing, turning, vulva location, spicule insertion and sperm transfer. The complexity of this behaviour is reflected in the sexually dimorphic anatomy and nervous system. Behavioural functions have been assigned to most of the male-specific sensory neurons by means of cell ablations; for example, the hook sensory neurons HOA and HOB are specifically required for vulva location. We have investigated how sensory perception of the hermaphrodite by the C. elegans male controls mating behaviours. Here we identify a gene, lov-1 (for location of vulva), that is required for two male sensory behaviours: response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine-threonine-rich amino terminus followed by two blocks of homology to human polycystins, products of the autosomal dominant polycystic kidney-disease loci PKD1 and PKD2. LOV-1 is the closest C. elegans homologue of PKD1. lov-1 is expressed in adult males in sensory neurons of the rays, hook and head, which mediate response, vulva location, and potentially chemotaxis to hermaphrodites, respectively. PKD-2, the C. elegans homologue of PKD2, is localized to the same neurons as LOV-1, suggesting that they function in the same pathway.
- Wilson PD, Geng L, Li X, Burrow CR
- The PKD1 gene product, "polycystin-1," is a tyrosine-phosphorylated protein that colocalizes with alpha2beta1-integrin in focal clusters in adherent renal epithelia.
- Lab Invest. 1999; 79: 1311-23
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Mutations in the PKD1 gene are responsible for autosomal dominant polycystic kidney disease (ADPKD). Although PKD1 has been cloned and shown to be expressed at high levels in the fetal ureteric bud and ADPKD cystic epithelia in the human kidney, the function of its encoded protein, "polycystin-1" is unknown. In this study we used primary and immortalized human renal epithelial cell lines derived from normal fetal, adult, and ADPKD kidneys, that endogenously express PKD1, to study the biologic function of the polycystin-1 protein. ADPKD renal epithelial cells expressed high levels of polycystin-1 protein and showed increased adhesion to type I collagen by comparison with normal adult human renal epithelia that expressed little polycystin. Adherent ADPKD cells also expressed high levels of alpha2beta1-integrin and their attachment was inhibited by a functional monoclonal antibody to alpha2-integrin. Double labeling and confocal microscopy as well as coimmunoprecipitation analysis showed overlapping colocalization of polycystin-1 with alpha2beta1-integrin as well as with the focal adhesion proteins vinculin and paxillin in multiprotein clusters localized to focal areas of cell membrane contact with type I collagen matrix after short periods of attachment. Immunoprecipitation and Western immunoblot studies also showed that polycystin-1 was posttranslationally modified by tyrosine phosphorylation. These studies suggest that the PKD1-encoded protein is part of a large multiprotein complex in epithelial cells that functions in the regulation of extracellular matrix interactions with the plasma membrane and cell cytoskeleton.
- Hateboer N et al.
- Comparison of phenotypes of polycystic kidney disease types 1 and 2. European PKD1-PKD2 Study Group.
- Lancet. 1999; 353: 103-7
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BACKGROUND: Although autosomal dominant polycystic kidney disease type 2 (PKD2) is known to have a milder clinical phenotype than PKD1, neither disorder has been compared with an unaffected control population in terms of survival. We report the findings of a multicentre survey that aimed to define more precisely the survival and clinical expression of PKD1 and PKD2. METHODS: Clinical data from 333 people with PKD1 (31 families) were compared with data from 291 people with PKD2 (31 families) and 398 geographically matched controls. Survival analysis was used to compare age-at-event data. Differences in the prevalence of complications were assessed by logistic regression. FINDINGS: Median age at death or onset of end-stage renal disease was 53.0 years (95% CI 51.2-54.8) in individuals with PKD1, 69.1 years (66.9-71.3) in those with PKD2, and 78.0 years (73.8-82.2) in controls. Women with PKD2 had a significantly longer median survival than men (71.0 [67.4-74.8] vs 67.3 [64.9-69.7] years), but no sex influence was apparent in PKD1. Age at presentation with kidney failure was later in PKD2 than in PKD1 (median age 74.0 [67.2-80.8] vs 54.3 [52.7-55.9] years). PKD2 patients were less likely to have hypertension (odds ratio 0.25 [95% CI 0.15-0.42]), a history of urinary-tract infection (0.50 [0.31-0.83]), or haematuria (0.59 [0.35-0.98]). INTERPRETATION: Although PKD2 is clinically milder than PKD1, it has a deleterious impact on overall life expectancy and cannot be regarded as a benign disorder.
- Watson P, Davey J
- Characterization of the Prk1 protein kinase from Schizosaccharomyces pombe.
- Yeast. 1998; 14: 485-92
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We report the isolation and characterization of a protein kinase from the fission yeast Schizosaccharomyces pombe. The proposed Prk1 protein contains 352 amino acids and has significant homology to the Ume5p kinase (also known as Srb10p, Ssn3p and Are1p) of the budding yeast Saccharomyces cerevisiae, a cyclin-dependent kinase involved in regulating the transcription of a diverse set of genes. Disruption of the prk1 gene increases flocculation but does not appear to have any other significant effect on cell behaviour. This defect can be overcome by expressing the UME5 gene, indicating that Prk1 is the fission yeast homologue of Ume5p.
- Hermand D et al.
- Fission yeast Csk1 is a CAK-activating kinase (CAKAK).
- EMBO J. 1998; 17: 7230-8
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Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).
- Wang Y, Dimitrov K, Garrity LK, Sazer S, Beverley SM
- Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant.
- Mol Biochem Parasitol. 1998; 96: 139-50
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Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many organisms. Alternating phases of growth, arrest, and differentiation are characteristics of the infectious cycle of many trypanosomatid parasites, raising the possibility that members of the trypanosomatid CRK gene family participate in the regulation of these essential processes. Here we describe properties of the CRK3 gene from Leishmania major, which encodes a 36 kDa protein kinase showing 60% amino acid sequence identity with human CDK2, including several conserved sites implicated in regulation of kinase activity. CRK3 mRNA was constitutively expressed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in stationary-phase promastigotes. When integrated into the genome and expressed at the optimal level, CRK3 was able to rescue the growth defect of a Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)), indicating that CRK3 is a functional homolog of cdc2. Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S. pombe mutant. This is the first example of functional expression of a trypanosomatid CRK in yeast, opening the way for further genetic studies within this amenable organism.
- Groenen PM, Vanderlinden G, Devriendt K, Fryns JP, Van de Ven WJ
- Rearrangement of the human CDC5L gene by a t(6;19)(p21;q13.1) in a patient with multicystic renal dysplasia.
- Genomics. 1998; 49: 218-29
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Genetic studies have implicated the short arm of chromosome 6 in congenital hydronephrosis. In previous studies, we described a fetus carrying a t(6;19)(p21;q13.1) as the sole cytogenetic anomaly and suffering from bilateral multicystic renal dysplasia caused by a bilateral complete pelviureteric junction obstruction, resulting in a massive hydronephrosis. Characterization of the chromosome 19 breakpoint region revealed that the transcription factor-encoding USF2 gene is affected. In this report, we show that the CDC5L gene on chromosome 6p is rearranged in the cells of the fetus. CDC5L encodes a protein that is related to the product of the Schizosaccharomyces pombe Cdc5 gene, which exerts its effects at the G2/M transition during cell cycle progression. We have established the genomic organization of the CDC5L gene and found that it consists of at least 16 exons spanning approximately 50 kb of chromosome segment 6p21. Northern blot analysis indicated that the gene is ubiquitously expressed as a single mRNA of about 3.4 kb in both fetal and adult tissues. The translation product of the CDC5L gene has an electrophoretic mobility of about 100 kDa and is predicted to be a nuclear protein, since it contains a Myb-related DNA binding domain and potential nuclear localization signals in its aminoterminal region. Immunocytochemical analysis confirmed the nuclear localization of the CDC5L protein. CDC5L was also predicted to contain a hydrophilic, proline-rich region in its central part, which might function as a transcriptional activating domain. The chromosome 6 breakpoint was found in the intron between exons 9 and 10, indicating that, as a direct result of the 6;19 translocation, the Myb-related DNA binding domains and the nuclear localization signals are separated from the putative transactivating domain. Northern blot and RT-PCR experiments revealed that the other CDC5L allele is unaffected, and in Western blot experiments, expression of the 100-kDa protein was detected in fibroblasts of the fetus. Expression of a truncated or hybrid CDC5L transcript resulting from the CDC5L rearrangement could not be demonstrated.
- Longtine MS, Fares H, Pringle JR
- Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function.
- J Cell Biol. 1998; 143: 719-36
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To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).
- Nakano K, Arai R, Mabuchi I
- 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.
- Genes Cells. 1997; 2: 679-94
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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.
- Toda T et al.
- The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.
- Mol Cell Biol. 1996; 16: 6752-64
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We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.
- Gilbreth M et al.
- The highly conserved skb1 gene encodes a protein that interacts with Shk1, a fission yeast Ste20/PAK homolog.
- Proc Natl Acad Sci U S A. 1996; 93: 13802-7
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The Shk1 protein kinase, a homolog of Saccharomyces cerevisiae Ste20 and mammalian p21Cdc42/Rac-activated kinases, is an essential component of a Ras- and Cdc42-dependent signaling cascade required for cell viability, normal morphology, and mitogen-activated protein kinase-mediated sexual responses in the fission yeast, Schizosaccharomyces pombe. To identify S. pombe proteins that modulate or mediate Shk1 functions, we conducted a two-hybrid screen for Shk1-interacting proteins. One of the genes identified as a result of this screen was skb1. We show that Skb1 interacts with a region of the N-terminal regulatory domain of Shk1 distinct from that to which Cdc42 binds, and that Shk1, Cdc42, and Skb1 are able to form a ternary complex in vivo. S.pombe cells carrying an skb1 null mutation are less elongate in morphology than wild-type cells and exhibit a moderate growth defect. The morphology defect of the skb1 deletion mutant is suppressed by overexpression of Shk1. Overexpression of Skb1 causes wild-type S. pombe cells to become hyperelongated. Additional genetic analyses described herein suggest that Skb1 is a component of the morphology control branch of the Ras signaling cascade in S. pombe and that it positively modulates Shk1 function. Homologs of Skb1 are encoded by open reading frames in the genomes of S. cerevisiae and Caenorhabditis elegans and by an uncharacterized human cDNA sequence. Thus, skb1 may be the first well-characterized member of a highly conserved family of genes encoding potential p21Cdc42/Rac-activated kinase regulators.
- Matsusaka T, Hirata D, Yanagida M, Toda T
- A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast.
- EMBO J. 1995; 14: 3325-38
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Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants. Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2. Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA. Interestingly, these mutant cells grew only in a monopolar manner. At a specific point in the G2 phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar. This change coincides with the distribution of cortical actin from one end of the cell to both ends. In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1+ encodes a novel protein kinase. Ectopic overexpression of ssp1+ resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells. Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction. These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases.
- Woo D
- Apoptosis and loss of renal tissue in polycystic kidney diseases.
- N Engl J Med. 1995; 333: 18-25
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BACKGROUND: Polycystic kidney disease is characterized by the enlargement of renal cysts, interstitial fibrosis, and gradual loss of normal renal tissue in association with progressive deterioration of renal function. The process causing the progressive loss of renal tissue is unknown, but it could be the result of a form of programmed cell death known as apoptosis. METHODS: We assayed apoptotic DNA fragmentation in normal and polycystic kidneys biochemically by gel electrophoresis and histochemically by in situ end-labeling. A DNA-specific dye, Hoechst 33258, was used to detect morphologic apoptosis in renal samples from patients with normal kidneys, polycystic kidney disease, and other kidney diseases. RESULTS: Apoptotic DNA fragmentation was detected in polycystic kidneys from 5 patients without renal failure and 11 patients with renal failure but not in kidneys from 12 patients with no renal disease. In situ end-labeling revealed apoptotic cells in glomeruli, in cyst walls, and in both cystic and noncystic tubules of the polycystic kidneys. No tubular apoptosis was detected in renal-biopsy specimens from five patients with IgA nephropathy, three patients with nephrosclerosis, two patients with focal glomerulosclerosis, one patient with diabetic nephropathy, six patients with acute tubular necrosis, or four patients with acute and four patients with chronic renal-transplant rejection. The capacity of polycystic kidney cells to undergo apoptosis was retained in vitro in the absence of uremia, ischemia, and other confounding pathologic conditions. CONCLUSIONS: Apoptotic loss of renal tissue may be associated with the progressive deterioration of renal function that occurs in patients with polycystic kidney disease.
- Nadasdy T, Laszik Z, Lajoie G, Blick KE, Wheeler DE, Silva FG
- Proliferative activity of cyst epithelium in human renal cystic diseases.
- J Am Soc Nephrol. 1995; 5: 1462-8
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Increased proliferative activity of the renal tubular epithelium is thought to be a prerequisite for renal cyst formation by many investigators. However, in humans, the exact in vivo proliferation rate of epithelial cells lining these cysts is not known. In this study, which used immunohistochemical methods with an antibody to proliferating cell nuclear antigen (PCNA), the proliferation index (PI) (percentage of PCNA positive cell nuclei among epithelial cells lining the renal cysts) was determined in 10 cases of autosomal dominant polycystic kidney disease (ADPKD), 8 cases of autosomal recessive polycystic kidney disease (ARPKD), and 8 cases of acquired cystic kidney disease (ACKD). Cysts with proximal and distal nephron phenotype and cysts with markedly thickened basement membranes, as well as cysts lined by atrophic (flattened), "regular" (cuboidal or cylindrical), and hyperplastic epithelium, were evaluated separately. The overall PI of cyst epithelium (excluding hyperplastic cysts) was 2.58 in ADPKD, was 10.5 in ARPKD, and was 3.61 in ACKD. Overall, there were only minor differences in the PI between the various types of cysts. Cysts with hyperplastic epithelium in ACKD (unlike in ADPKD) showed a high PI (9.1). For comparison, the PI of two renal cell carcinomas occurring in two ACKD cases was also determined (13.70 and 8.67%). The PI of tubular epithelium in normal kidneys was only 0.22 to 0.33%, depending on the tubule segment. In contrast, in polycystic kidneys, those noncystic segments of the nephron from which the cysts are thought to originate (distal nephron (specifically collecting duct)) in ARPKD, primarily distal in ADPKD, proximal and distal in ACKD, had PI values similar to those of the cyst epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)
- Gabow PA
- Autosomal dominant polycystic kidney disease.
- N Engl J Med. 1993; 329: 332-42
- Toda T, Shimanuki M, Yanagida M
- Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases.
- Genes Dev. 1991; 5: 60-73
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Staurosporine, a potent inhibitor of protein kinase C, arrests fission yeast cell elongation specifically at a stage immediately after cell division. We isolated two genes, which, when carried on multicopy plasmids, confer drug resistance in fission yeast. One, spk1+, encodes a protein kinase highly similar (54% identity) to those encoded by the mammalian ERK1/MAP2 kinase and the budding yeast KSS1 and FUS3 genes. It is not essential for vegetative growth of Schizosaccharomyces pombe cells but is required for conjugation. The spk1+ gene product is a 45-kD protein enriched in the nucleus, and its level increases 10-fold after addition of staurosporine. The other gene pap1+ encodes an AP-1-like transcription factor that contains a region rich in basic amino acids followed by a "leucine zipper" motif. The pap1+ gene is required for spk1(+)-conferred staurosporine resistance. These two genes appear to function as a part of the fission yeast growth control pathway.
- Reeders ST et al.
- A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16.
- Nature. 1985; 317: 542-4
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Adult polycystic kidney disease (APCKD) is a common and often lethal multi-organ disease with an autosomal dominant pattern of inheritance; approximately 1 in 1,000 people carry the mutant gene. The major pathological abnormality is the development and progressive enlargement of cysts in several organs including the liver, pancreas and spleen as well as the kidneys. The basic biochemical defect which leads to the formation of cysts remains unknown. Cyst development, which is not retarded by any known therapy, leads to irreversible renal failure and death at a mean age of 51 unless dialysis or transplantation are used. Patients with the disease account for 9% of chronic dialysis requirement. The first symptoms tend to occur in the fourth decade, after most patients have reproduced. Presymptomatic diagnosis depends on the ultrasonographic detection of cysts, but exclusion cannot be achieved by this means; 34% of at-risk patients in the second decade and 14% in the third will go on to develop cysts after negative diagnosis. The low sensitivity of diagnostic techniques in this critical age-range imposes severe limitations on genetic counselling and the condition cannot be identified prenatally. Hence we have searched for a linkage marker for APCKD; we show here that the APCKD locus is closely linked to the alpha-globin locus on the short arm of chromosome 16 (zeta = 25.85, theta = 0.05).