Stress-activated protein kinase pathway in cell cycle control of fission yeast.
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Amino Acid Sequence↗
Biomedical subjects
K Shiozaki
Publications and source records attributed to K Shiozaki.
Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade.
Spc1 in Schizosaccharomyces pombe is a member of the stress-activated protein kinase family, an evolutionary conserved subfamily of mitogen-activated protein kinases (MAPKs). Spc1 is activated by a MAPK kinase homologue, Wis1, and negatively regulated by Pyp1 and Pyp2 tyrosine phosphatases. Mutations in the spc1+ and wis1+ genes cause a G2 cell cycle delay that is exacerbated during stress. Herein, we describe two upstream regulators of the Wis1-Spc1 cascade. wik1+ (Wis1 kinase) was identified from its homology to budding yeast SSK2, which encodes a MAPKK kinase that regulates the HOG1 osmosensing pathway. Delta wik1 cells are impaired in stress-induced activation of Spc1 and show a G2 cell cycle delay and osmosensitive growth. Moreover, overproduction of a constitutively active form of Wik1 induces hyperactivation of Spc1 in wis1(+)-dependent manner, suggesting that Wik1 regulates Spc1 through activation of Wis1. A mutation of mcs4+ (mitotic catastrophe suppressor) was originally isolated as a suppressor of the mitotic catastrophe phenotype of a cdc2-3w wee1-50 double mutant. We have found that mcs4- cells are defective at activation of Spc1 in response to various forms of stress. Epistasis analysis has placed Mcs4-upstream of Wik1 in the Spc1 activation cascade. These results indicate that Mcs4 is part of a sensor system for multiple environmental signals that modulates the timing of entry into mitosis by regulating the Wik1-Wis1-Spc1 kinase cascade. Inactivation of the sensor system delays the onset of mitosis and rescues lethal premature mitosis in cdc2-3w wee1-50 cells.
Amino Acid Sequence↗
[A case of malignant hemangioendothelioma of the lung with rapid growth of pulmonary metastasis].
Malignant hemangioendothelioma of the lung is very rare and difficult to diagnose. The growth is rapid and the prognosis is very unfavorable. We report an operated case of the disease with rapid growth of pulmonary metastasis. The silver, vimentin and factor VIII stainings were available for diagnosis of this disease. The 46-year-old female patient died of bleeding from the pulmonary metastasis eight months after the operation without other distant metastasis. Even if a lung tumor is pointed out and thought to be benign, frequent follow up is necessary. Furthermore, we recommend positive lung biopsy by thoracotomy or video-assisted-thoracic-surgery.
Female↗
[Hepatic arterial chemotherapy for liver cancer over a period of 8 years].
Hepatic arterial chemotherapy was performed for 27 patients with primary (3), metastatic liver cancer (21), and 3 other cases, over a period of 8 years. Chemotherapy was performed by intermittent hepatic arterial infusion of 5-FU or FAM (in case of metastatic tumor from colorectal cancer), FAM (from gastric cancer), and CDDP or Farmorubicin (HCC). Hepatic resection was performed in 10 cases of metastatic tumor from colorectal cancer, and 8 cases of 10 were curative operation. The 5-year survival rates of curative liver resection group, and non-curative liver resection or non-resection group were 57.1% and 12.5%, respectively. As is the case with metastatic cancer from gastric cancer, pancreatic cancer, and hepatocellular carcinoma (HCC), the prognosis was poor except for one CR case of HCC. We concluded that hepatic arterial chemotherapy may be recommended for a curative resected case of liver metastasis from colorectal cancer.
Antineoplastic Combined Chemotherapy Protocols↗
[A case of liver metastasis of gastric leiomyosarcoma successfully treated by transarterial hepatic chemo-embolization and intra hepato-arterial chemotherapy repeated with infusion-a-port].
The patient was a 68-year-old male, who underwent total gastrectomy for giant leiomyosarcoma of the stomach and then had multiple hepatic metastases one year and six months later. Thus, transarterial hepatic chemo-embolization therapy with Lipiodol, adriamycin and gelfoam was given. Moreover, using a reservoir catheter and infusion arterial port, intermittent arterial infusion therapy with adriamycin, cyclophosphamide, and vincristine was attempted. In the metastasis lesion where there were rich blood vessels, Lipiodol was accumulated and the tumor was reduced on abdominal CT. The result indicated the efficacy of this treatment.
Aged↗
Conjugation, meiosis, and the osmotic stress response are regulated by Spc1 kinase through Atf1 transcription factor in fission yeast.
The stress-activated Wis1-Spc1 protein kinase cascade links mitotic control with environmental signals in Schizosaccharomyces pombe. Fission yeast spc1- mutants are delayed in G2 during normal growth and undergo G2 arrest when exposed to osmotic or oxidative stress. Here we report that Spc1 also has an important role in regulating sexual development in S. pombe. This discovery arose from the observation that Spc1 is activated in response to nitrogen limitation, a key signal that promotes conjugation in fission yeast. Mutant spc1- cells are defective at arresting in G2 during nitrogen starvation and exhibit a poor mating ability. These deficiencies correlate with a failure to induce transcription of ste11+, a gene that encodes a transcription factor responsible for expression of various meiotic genes. Two genes, atf1+ and atf21+, were cloned as multicopy suppressors of the spc1- mating defect. Atf1 and Atf21 are bZIP transcription factors that are most closely related to human ATF-2/CRE-BP1. Spc1 is required for stress-induced phosphorylation of Atf1. Atf1 is required for induction of meiotic genes and stress-response genes, such as gpd1+ and pyp2+, that are transcriptionally regulated by Spc1. atf1- and spc1- mutants are sensitive to osmotic stress and impaired for sexual development, showing that fission yeast uses a common pathway to respond to cytotoxic stress and nitrogen starvation. However, unlike spc1- mutants, atf1- cells have no mitotic cell-cycle defect, indicating that the stress response pathway bifurcates at Spc1 to regulate independently meiosis and mitosis.
Activating Transcription Factor 1↗
Phosphorylation of human m1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 and protein kinase C.
Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) were expressed in and purified from insect Sf9 cells and then subjected to phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase C purified from rat brain (a mixture of alpha, beta, and gamma types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein beta gamma subunits stimulated the phosphorylation by GRK2 but did not affect the phosphorylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phoshorylation by GRK2 and PKC, respectively. The number of incorporated phosphates was 7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by GRK2. Major sites phosphorylated by GRK2 and PKC were located in the third intracellular loop and the carboxyl-terminal tail, respectively. These results indicate that GRK2 and PKC phosphorylate different sites of m1 receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound receptors.
Amino Acid Sequence↗
Selective Isolation of DNA or RNA Using Single-Stranded DNA Affinity Latex Particles.
We developed latex particles with a diameter of about 0.22 µm on which single-stranded (ss) DNA was covalently coupled to select or enrich its complementary DNA or mRNA. DNA was first covalently coupled to the latex particles in the double-stranded (ds) form with both blunt and protruding ends. More than 80% of the dsDNA was coupled through the ssDNA stretch at its protruding end. The presence of NaCl in the immobilization reaction severely inhibited DNA from coupling to the particles. The particles were then treated with alkali or heated to denature the dsDNA and sedimented by a brief centrifugation to yield ssDNA immobilized particles. They allowed the selective and efficient isolation of a desired RNA from total cellular RNA.
Journal Article↗
Spatial organization of the Nim1-Wee1-Cdc2 mitotic control network in Schizosaccharomyces pombe.
In Schizosaccharomyces pombe the onset of mitosis is regulated by a network of protein kinases and phosphatases. The M-phase inducing Cdc2-Cdc13 cyclin-dependent kinase is inhibited by Wee1 tyrosine kinase and activated by Cdc25 phosphatase. Wee1 is negatively regulated by Nim1 protein kinase. Here, we describe investigations aimed at better understanding the role of Nim1 in the mitotic control. The most important finding to emerge from these studies is that Wee1 and Nim1 have different patterns of intracellular localization. Immunofluorescence confocal microscopy has revealed that Nim1 is localized in the cytoplasm, whereas it substrate Wee1 is predominantly localized in the nucleus. Previous studies showed that the Cdc2-Cdc13 complex is located in the nucleus. Diversion of Nim1 to the nucleus, accomplished by addition of the SV40 nuclear localization signal, caused the advancement of M, confirming that Nim1 has restricted access to Wee1 in vivo. We propose that the intracellular distribution of Nim1 and Wee1 may serve to coordinate the regulation of nuclear Cdc2-Cdc13 with cytoplasmic growth.
CDC2 Protein Kinase↗
Activation and regulation of the Spc1 stress-activated protein kinase in Schizosaccharomyces pombe.
Spc1, an osmotic-stress-stimulated mitogen-activated protein kinase (MAPK) homolog in the fission yeast Schizosaccharomyces pombe, is required for the induction of mitosis and survival in high-osmolarity conditions. Spc1, also known as Sty1, is activated by Wis1 MAPK kinase and inhibited by Pyp1 tyrosine phosphatase. Spc1 is most closely related to Saccharomyces cerevisiae Hog1 and mammalian p38 kinases. Whereas Hog1 is specifically responsive to osmotic stress, we report here that Spc1 is activated by multiple forms of stress, including high temperature and oxidative stress. In this regard Spc1 is more similar to mammalian p38. Activation of Spc1 is crucial for survival of various forms of stress. Spc1 regulates expression of genes encoding stress-related proteins such as glycerol-3-phosphate dehydrogenase (gpd1+) and trehalose-6-phosphate synthase (tps1+). Spc1 also promotes expression of pyp2+, which encodes a tyrosine phosphatase postulated as a negative regulator of Spc1. This proposal is supported by the finding that Spc1 associates with Pyp2 in vivo and that the amount of Spc1 tyrosine phosphorylation is lower in a Pyp2-overproducing strain than in the wild type. Moreover, the level of stress-stimulated gpd1+ expression is higher in delta pyp2 mutants than in the wild type. These findings demonstrate that Spc1 promotes expression of genes involved in stress survival and that of regulation may be commonly employed to modulate MAPK signal transduction pathways in eukaryotic species.
Animals↗
Pneumonitis during interferon and/or herbal drug therapy in patients with chronic active hepatitis.
We report four cases of acute pneumonitis due either to interferon, or a herbal drug, "Sho-saiko-to", or both in combination, in patients with chronic active hepatitis, focusing on its pathogenesis and response to prednisolone therapy. These cases shared common clinical features: fever, dry cough, dyspnoea, hypoxaemia, diffuse infiltrates both on chest radiography and chest computed tomography, restrictive pulmonary functional impairment, and alveolitis on examination of transbronchial lung biopsy, all of which suggest acute interstitial pneumonia. Furthermore, lymphocytosis was observed in association with the dominant CD8+ T-cell subset in bronchoalveolar lavage fluid. A lymphocyte stimulation test using peripheral blood was positive to interferon in one case and to Sho-saiko-to in another. All patients responded to oral prednisolone therapy. Peripheral soluble interleukin-2 receptor levels decreased in parallel with improvement in the clinical course. All patients were free of symptoms with a follow-up of 1-3 yrs. We conclude that interferon- and/or Sho-saiko-to-induced acute pneumonitis may be due to allergic-immunological mechanisms rather than toxicity, and that peripheral levels of soluble interleukin-2 receptor appear to be good markers of disease activity.
Adult↗
Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast.
In fission yeast the onset of mitosis is brought about by Cdc2/Cdc13 kinase, which is inhibited by the Wee1/Mik1 tyrosine kinases and activated by Cdc25 tyrosine phosphatase. This control network integrates many signals, including those that monitor DNA replication, DNA damage and cell size. We report here that a fission yeast MAP kinase pathway links the cell-cycle G2/M control with changes in the extracellular environment that affect cell physiology. Fission yeast spc1- mutants have a G2 delay that is greatly exacerbated by growth in high osmolarity media and nutrient limitation. A lethal interaction of spc1 and cdc25 mutations shows that Spc1 promotes the onset of mitosis. Spc1 is a MAP kinase homologue that is activated by Wis1 kinase in response to osmotic stress and nutrient limitation. Spc1 is inactivated by Pyp1, a phosphatase previously identified as a mitotic inhibitor. Pyp1 dephosphorylates only tyrosine-173 of Spc1, unlike the dual-specificity phosphatases that have been shown to regulate other MAP kinases.
Amino Acid Sequence↗
Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast.
With the goal of discovering the cellular functions of type 2C protein phosphatases, we have cloned and analyzed two ptc (phosphatase two C) genes, ptc2+ and ptc3+, from the fission yeast Schizosaccharomyces pombe. Together with the previously identified ptc1+ gene, the enzymes encoded by these genes account for approximately 90% of the measurable PP2C activity in fission yeast cells. No obvious growth defects result from individual disruptions of ptc genes, but a delta ptc1 delta ptc3 double mutant displays aberrant cell morphology and temperature-sensitive cell lysis that is further accentuated in a delta ptc1 delta ptc2 delta ptc3 triple mutant. These phenotypes are almost completely suppressed by the presence of osmotic stabilizers, strongly indicating that PP2C has an important role in osmoregulation. Genetic suppression of delta ptc1 delta ptc3 lethality identified two loci, mutations of which render cells hypersensitive to high-osmolarity media. One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog. The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation. These data indicate that divergent yeasts have functionally conserved MAP kinase pathways, which are required to increase intracellular osmotic concentrations in response to osmotic stress. Moreover, our observations implicate PP2C enzymes as also having an important role in signal transduction processes involved in osmoregulation, probably acting to negatively regulate the osmosensing signal that is transmitted through Wis1 MAP kinase kinase.
Amino Acid Sequence↗
Protein phosphatase 2C, encoded by ptc1+, is important in the heat shock response of Schizosaccharomyces pombe.
Protein phosphatase 2C (PP2C), an Mg(2+)-dependent enzyme that dephosphorylates serine and threonine residues, defines one of the three major families of structurally unrelated eukaryotic protein phosphatases. Members of the two other families of protein phosphatases are known to have important cellular roles, but very little is known about the biological functions of PP2C. In this report we describe a genetic investigation of a PP2C enzyme in the fission yeast Schizosaccharomyces pombe. We discovered ptc1+ (phosphatase two C) as a multicopy suppressor gene of swo1-26, a temperature-sensitive mutation of a gene encoding the heat shock protein hsp90. The ptc1+ gene product is a 40-kDa protein with approximately 24% identity to a rat PP2C protein. Purified Ptc1 has Mg(2+)-dependent casein phosphatase activity, confirming that it is a PP2C enzyme. A ptc1 deletion mutant is viable and has approximately normal levels of PP2C activity, observations consistent with the fact that ptc1+ is a member of a multigene family. Although a ptc1 deletion mutant is viable, it has a greatly reduced ability to survive brief exposure to elevated temperature. Moreover, ptc1+ mRNA levels increase 5- to 10-fold during heat shock. These data, demonstrating that Ptc1 activity is important for survival of heat shock, provide one of the first genetic clues as to the biological functions of PP2C.
Amino Acid Sequence↗
Cellular function of protein phosphatase 2C in yeast.
Protein phosphatase 2C (PP2C) is one of four major classes of protein serine/threonine-phosphatases, which requires Mg2+ for its activity. PP2C activity distributes ubiquitously among eukaryotes and enzymes purified from mammalian tissues were well characterized biochemically, however, very little is known about their biological function. We have been trying to identify cellular function of PP2C by genetic analyses in fission yeast Schizosaccharomyces pombe. So far, three PP2C homologs, ptc1+, ptc2+, and ptc3+, have been cloned and their activity were detected in the S. pombe cell lysates. Experiments using ptc mutants constructed by gene disruption technique have revealed the involvement of PP2C in the heat shock response and possibly osmoregulation in yeast cells.
Cloning, Molecular↗
[The appearance and enlargement of localized pulmonary granuloma with eosinophilic infiltration during tuberculosis chemotherapy].
A pulmonary tumorous shadow appeared and enlarged in a 25 years-old male patient undergoing intensive chemotherapy for tuberculosis. The chest X-rays taken on admission revealed effused pleura in the right lung and nodular shadows in the upper area of the right lung. After 40 days of using isoniazid (INH), rifampicin (RFP) and streptomycin (SM), a homogeneous opacity, not previously observed, appeared in the middle area of the right lung (S5). Microscopic examination of the tissues obtained during a transbronchial lung biopsy disclosed epithelioid cell granulomas with marked eosinophilic infiltration. The presence of eosinophilic infiltration due to the admission of antituberculosis agents was disregarded because no change was observed in the new granulomatous shadows during the drug challenge tests and the lymphocyte stimulation test to INH, RFP and SM was negative. Transient aggravation during the initial phase of chemotherapy for pulmonary tuberculosis, such as in this case, is suspected cause by some eosinophilic allergic-induced mechanisms, against bacillary components.
Adult↗
Effects of magnesium ion on the interaction of atrial muscarinic acetylcholine receptors and GTP-binding regulatory proteins.
Muscarinic acetylcholine receptors (mAChR) purified from porcine atrium were reconstituted into lipid vesicles with GTP-binding regulatory proteins (G proteins, Gi, Go, or Gn) purified from porcine cerebrum. Apparent affinities of the reconstituted mAChR and G proteins for carbachol and GDP, respectively, were estimated from the effects of these ligands on the binding of [3H]-L-quinuclidinyl benzilate ([3H]QNB) to mAChR and [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) to G proteins in the presence of different concentrations of MgCl2. A total of 30-35% of reconstituted mAChRs exhibited low affinity for carbamylcholine, irrespective of the presence or absence of guanine nucleotides, and the remainder of the mAChRs showed high affinities for carbamylcholine in the absence of GTP or GDP and a low affinity in their presence. The affinity for carbamylcholine in the absence of guanine nucleotides, but not in their presence, increased with increases in MgCl2 concentration. Apparent Kd's for carbamylcholine were estimated to be approximately 100 microM in the presence of guanine nucleotides, 1.5 microM in the absence of guanine nucleotide and Mg2+ (< 0.1 microM), and 0.1 microM in the absence of guanine nucleotide and the presence of MgCl2 (10 mM). These results indicate that mAChRs may assume at least three different conformations that are characterized by different affinities for agonists. Furthermore, the data suggest that MgCl2 is not necessary for the formation of the mAChR-G protein complex, but can induce a conformational change in the complex. On the other hand, the presence of MgCl2 was necessary for carbamylcholine to influence the binding of guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗