Pleckstrin homology domain interacts with Rkp1/Cpc2, a RACK1 homolog, to modulate Pck2-mediated signaling process in Schizosaccharomyces pombe.

Rkp1/Cpc2, a fission yeast RACK1 homolog, interacts with Pck2, a PKC homolog, and is involved in the regulation of pck2-mediated signaling process. The N-terminal region of split pleckstrin homology domain (nPH) in human PLC-gamma1 bound to Rkp1/Cpc2 concomitantly with Pck2. nPH inhibited kinase activity of GST-Pck2 purified from Schizosaccharomyces pombe in vitro. The lethality induced by pck2(+) overexpression was suppressed by coexpression of either rkp1(+) or nPH domain. This result suggests that Rkp1/Cpc2 interacts with PH domain-containing protein and regulates the Pck2-mediated signaling process in S. pombe.

Peripheral administration of an angiotensin II AT(1) receptor antagonist decreases the hypothalamic-pituitary-adrenal response to isolation Stress.

Angiotensin II, which stimulates AT(1) receptors, is a brain and peripheral stress hormone. We pretreated rats with the AT(1) receptor antagonist candesartan for 13 d via sc-implanted osmotic minipumps, followed by 24-h isolation in individual metabolic cages. We measured angiotensin II receptor-type binding and mRNAs and tyrosine hydroxylase mRNA by quantitative autoradiography and in situ hybridization, catecholamines by HPLC, and hormones by RIA. Isolation increased AT(1) receptor binding in hypothalamic paraventricular nucleus as well as anterior pituitary ACTH, and decreased posterior pituitary AVP. Isolation stress also increased AT(1) receptor binding and AT(1B) mRNA in zona glomerulosa and AT(2) binding in adrenal medulla, adrenal catecholamines, tyrosine hydroxylase mRNA, aldosterone, and corticosterone. Candesartan blocked AT(1) binding in paraventricular nucleus and adrenal gland; prevented the isolation-induced alterations in pituitary ACTH and AVP and in adrenal corticosterone, aldosterone, and catecholamines; abolished the increase in AT(2) binding in adrenal medulla; and substantially decreased urinary AVP, corticosterone, aldosterone, and catecholamines during isolation. Peripheral pretreatment with an AT(1) receptor antagonist blocks brain and peripheral AT(1) receptors and inhibits the hypothalamic-pituitary-adrenal response to stress, suggesting a physiological role for peripheral and brain AT(1) receptors during stress and a possible beneficial effect of AT(1) antagonism in stress-related disorders.

Isolation of a novel gene from Schizosaccharomyces pombe: stm1+ encoding a seven-transmembrane loop protein that may couple with the heterotrimeric Galpha 2 protein, Gpa2.

A putative seven transmembrane protein gene, stm1(+), which is required for proper recognition of nitrogen starvation signals, was isolated as a multicopy suppressor of a ras1 synthetic lethal mutant in Schizosaccharomyces pombe. Under nitrogen-deficient conditions, transcription of the stm1 gene was induced; deletion of stm1 was associated with early entry into G(1) arrest. Under nutritionally sufficient conditions, overexpression of Stm1 inhibited vegetative cell growth, resulted in decreased intracellular cAMP levels, increased the expression of the meiosis-specific genes ste11, mei2, and mam2, and facilitated sexual development in homothallic cells. However inhibition of vegetative cell growth and reduction of cAMP levels were not observed in a deletion mutant of the heterotrimeric G protein Galpha2 gene, gpa2, that is responsible for regulating intracellular cAMP levels, a key factor in determining the sexual development in S. pombe. Stm1 protein was shown to interact with Gpa2 through its C-terminal transmembrane domains 5-7. Mutation at Lys(199) in the C-terminal domain (stm1(K199A)) abolished the Stm1 overexpression effect on lowering cAMP levels. Induction of ste11, a meiosis-specific gene transcription factor, by Stm1 overexpression was enhanced in gpa2-deleted cells but was absent in a deletion mutant of sty1, a key protein kinase that links mitotic control with environmental signals and induces stress-responsive genes. Moreover, deletion of both stm1 and ras1 caused delayed entry into G(1) arrest in S. pombe when the cells were grown in a nitrogen-deficient medium. Thus we consider that the stm1 gene can function through Gpa2-dependent and/or -independent pathways and may play a role in providing the prerequisite state for entering the pheromone-dependent differentiation cycle in which heterotrimeric Galpha1 protein, Gpa1, and Ras1 play major roles. Stm1 could function as a sentinel molecule sensing the nutritional state of the cells, stopping the proliferative cell cycle, and preparing the cell to enter meiosis under nutritionally deficient conditions.

Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling.

The Rkp1/Cpc2, a fission yeast RACK1 homolog, interacted with Pck2, one of the known PKC homologs, in vivo and in vitro. The rkp1-deletion mutants (Deltarkp1) are elongated and the pck2-deletion mutant (Deltapck2) showed abnormal morphology. The double-deletion mutant (Deltarkp1Deltapck2) showed more aberrant cell shapes and was sensitive to high salt concentration. Both Deltarkp1 and Deltapck2 cells were sensitive to latrunculin B (Lat B) which inhibits actin polymerization. The cells expressing the human RACK1 homolog complemented the latrunculin B sensitivity of Deltarkp1 indicating that human RACK1 is a functional homolog of Rkp1/Cpc2. We propose that Rkp1/Cpc2 may function as a receptor for Pck2 in the regulation of actin cytoskeleton organization during cell wall synthesis and morphogenesis of Schizosaccharomyces pombe.

Increased AT(1) receptor expression and mRNA in kidney glomeruli of AT(2) receptor gene-disrupted mice.

The proposed feedback between angiotensin II AT(2) and AT(1) receptors prompted us to study AT(1) receptor expression in kidneys of male AT(2) receptor-gene disrupted mice (agtr2 -/y). In wild-type (agtr2 +/y) mice, AT(1) receptor binding and mRNA is abundant in glomeruli, and AT(1) receptor binding is also high in the inner stripe of the outer medulla. AT(2) receptors are scarce, primarily associated to cortical vascular structures. In agtr2 -/y mice, AT(1) receptor binding and mRNA were increased in the kidney glomeruli, and AT(1) receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although AT(2) receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters AT(1) receptor expression. AT(1) upregulation alone may explain the AT(2) gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect AT(1)/AT(2) receptor feedback could have clinical significance because AT(1) antagonists are widely used in medical practice.

Gerbil angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development.

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT1 Angiotensin II receptors. The recently cloned gerbil AT1 receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT1 receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region. We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life. The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT1 receptors in the development and function of the corticohippocampal system.

DNA-induced conformational change of Gaf1, a novel GATA factor in Schizosaccharomyces pombe.

A novel GATA factor in Schizosaccharomyces pombe, Gaf1, containing one zinc-finger motif was studied for conformational change that was induced by DNA-binding. Gaf1 was shown to bind to the upstream activation sequence of a gene in Saccharomyces cerevisiae containing GATA element by gel mobility shift assay. Circular dichroism spectra of Gaf1 indicated an increase of alpha-helix content of Gaf1 occurred upon binding to the upstream activation sequence. These results suggest that the binding of Gaf1 to the GATA element is required for the conformational change that may precede transactivation of the target gene(s).

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata).

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.

Molecular cloning and pharmacological characterization of an atypical gerbil angiotensin II type-1 receptor and its mRNA expression in brain and peripheral tissues.

In the gerbil brain, most of the [125I]Sarcosine1-Angiotensin II binding sites are atypical, not sensitive to displacement with selective Angiotensin II AT1 and AT2 receptor ligands. A similar atypical binding profile exists in the gerbil kidney, where binding is highly expressed. We isolated a 2197 base pair clone from a gerbil kidney cDNA library which encodes a 359 amino acid protein with higher than 90% homology to other mammalian angiotensin II AT1 receptors. When expressed in COS-7 cells, stimulation by Angiotensin II of both the cloned gerbil receptor or the human AT1 receptor enhanced IP3 production to a similar degree. In COS-7 cells, the gerbil receptor also had a ligand affinity profile similar to that of the human AT1 receptor, but showed greatly reduced affinity for losartan (IC50=3480+/-174 nM). In the gerbil brain, in situ hybridization revealed receptor mRNA in circumventricular organs, selective hypothalamic, midbrain and brain stem areas, and in the hippocampus, where high mRNA expression was detected in the stratum pyramidale of the CA1 and CA2 subfields, and in the stratum granulosum of the dentate gyrus. The expression pattern of receptor mRNA corresponded well with that of atypical [125I]Sar1-Ang II binding. In situ hybridization and Southern blot experiments using riboprobes against the open reading frame and the 3'-untranslated region of the cloned gerbil Ang II receptor cDNA suggest that gerbils have, like other rodents, two AT1 receptor subtypes. The receptor mRNA distribution of the cloned gerbil Ang II receptor corresponds to the distribution of AT1A receptors described in other rodent species.

Molecular cloning of gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator.

As a first step to elucidate the functions of Schizosaccharomyces pombe (S. pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S. pombe. The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1. The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids. Disruption of this gene has no effect on cell viability and growth rate. The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S. cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain. These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.

Isolation of a novel heat shock protein 70-like gene, pss1+ of Schizosaccharomyces pombe homologous to hsp110/SSE subfamily.

A novel heat shock protein 70 (HSP70) gene, pss1+, of fission yeast, Schizosaccharomyces pombe (S. pombe), has been isolated as a multicopy suppressor of a synthetic lethal mutant of ras1+, which shows severe retardation of growth and aggregation phenotype when the ras1 gene function is absent. The pss1+ gene functionally complements the growth defect of the mutant. Sequence analysis revealed that pss1+ encodes an open reading frame (ORF) of 730amino acids that is homologous to the HSP70 family proteins. The Pss1 has high homology to the Saccharomyces cerevisiae (S. cerevisiae) heat shock protein Sse1p/Msi3p (43% identity) that belongs to the HSP110/SSE subfamily of HSP70. The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of pss1+ gene. The transcript level of pss1+ was moderately abundant during steady-state growth at 25 degrees C and increased a few-fold upon shifting to 42 degrees C. Furthermore, transcription of pss1+ increased in nitrogen-starved conditions. Disruption of the pss1+ gene confers a temperature-sensitive growth phenotype and unexpectedly causes the increase in thermotolerance in S. pombe.

Isolation of a new member of DnaJ-like heat shock protein 40 (Hsp40) from human liver.

A new member of Hsp40, HLJ1, consisting of 337 amino acids, was cloned from a human liver cDNA library. The deduced amino acid sequence of HLJ1 has an 84% homology (69% identity) with that of HDJ-1 isolated from human placenta. Northern analysis showed that expression of the HLJ1 gene is heat-inducible and its transcription shows some degree of preference in heart, skeletal muscle, and pancreas.

A novel protein, Psp1, essential for cell cycle progression of Schizosaccharomyces pombe is phosphorylated by Cdc2-Cdc13 upon entry into G0-like stationary phase of cell growth.

A novel gene, psp1(+), which functionally complements a temperature-sensitive mutant defective in cell cycle progression both in G1/S and G2/M has been isolated from the genomic and cDNA libraries of Schizosaccharomyces pombe. Disruption of this gene is lethal for cell growth at 30 degrees C indicating that it is an essential gene for vegetative cell growth. Western analysis of the protein by polyclonal antibody made from glutathione S-transferase-Psp1 fusion protein indicated that the Psp1 protein exists in two different molecular weight forms depending on the growth state of the cell. In vitro experiments with a phosphatase showed that this difference is due to phosphorylation. The dephosphorylated form of the protein is dominant in actively growing cells whereas the phosphorylated form becomes the major species when cells enter the stationary phase. The Cdc2-Cdc13 complex is shown to phosphorylate the GST-Psp1 fusion protein in vitro, and site-directed mutagenesis and phosphoamino acid analysis indicated that the serine residue at position 333 in the carboxyl-terminal region is required for phosphorylation. In situ fluorescein isothiocyanate-conjugated antibody staining showed that this protein tends to be localized to both ends of the cell upon entry into the stationary phase of cell growth. However, overexpression of the novel protein Psp1 in actively growing cells inhibits cell growth causing accumulation of DNA (4n or 8n). Thus we speculate that Psp1 can function at both G1/S and G2/M phases complementing the defect of the new mutant we have isolated. It is likely that Psp1 is required both for proper DNA replication and for the process of mitosis.

Molecular cloning of GAF2, a Schizosaccharomyces pombe GATA factor, which has two zinc-finger sequences.

By low stringency screening of a lambda-Shizosaccharomyces pombe genomic library, we have cloned a GATA factor homologous gene, gaf2+, within a 3.1-kb EcoRI fragment. The gaf2 ORF predicts a protein of M(r) 61 kDa consisting of intronless 564 amino acids corresponding to 1,692 bp. Gaf2 has two zinc-fingers as Urbs1 of Ustilago maydis, whereas most of fungal GATA factors have only one zinc-finger. The separation between two zinc-fingers of Gaf2 is rather long. In addition to gaf2, the sequence analysis revealed a Val-tRNA gene in the 3'-flanking region of gaf2. Northern blot analysis indicated that the gaf2 gene is transcribed constitutively irrespective of the nitrogen source in a medium.