The Rho-related protein Rnd1 inhibits Ca2+ sensitization of rat smooth muscle.
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1. The small GTP-binding Rho proteins are involved in the agonist-induced Ca2+ sensitization of smooth muscle. The action and the expression of Rnd1, a new member of the Rho protein family constitutively bound to GTP, has been studied in rat smooth muscle. 2. Recombinant prenylated Rnd1 (0.01-0.1 mg ml-1) dose dependently inhibited carbachol- and GTPgammaS-induced Ca2+ sensitization in beta-escin-permeabilized ileal smooth muscle strips but had no effect on the tension at submaximal [Ca2+] (pCa 6.3). Rnd1 inhibited GTPgammaS-induced tension without shifting the dose-response curves to GTPgammaS. 3. pCa-tension relationships were not modified by Rnd1 and the rise in tension induced through the inhibition of myosin light chain phosphatase by calyculin A was not affected by Rnd1. 4. The Ca2+ sensitization induced by recombinant RhoA was completely abolished when RhoA and Rnd1 were applied together. 5. Rnd1 was expressed at a low level in membrane fractions prepared from intestinal or arterial smooth muscles. The expression of Rnd1 was strongly increased in ileal and aortic smooth muscle from rats treated with progesterone or oestrogen. Progesterone-treated ileal muscle strips showed a decrease in agonist-induced Ca2+ sensitization. 6. The present study shows that (i) Rnd1 inhibits agonist- and GTPgammaS-induced Ca2+ sensitization of smooth muscle by specifically interfering with a RhoA-dependent mechanism and (ii) an increase in Rnd1 expression may account, at least in part, for the steroid-induced decrease in agonist-induced Ca2+ sensitization. (+info)
Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import.
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Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends upon preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Here we report that Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and that progesterone treatment abrogates this binding. We demonstrate that Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation. (+info)
Effects of microcystins on phosphorylase-a binding to phosphatase-2A: kinetic analysis by surface plasmon resonance biosensor.
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Cyclic heptapeptide microcystins are a group of hepatoxicants which exert the cytotoxic effects by inhibiting the catalytic activities of phosphatase-2A (PP-2A) and phosphatase-1 (PP-1) and thus disrupt the normal signal transduction pathways. Microcystins interact with PP-2A and PP-1 by a two-step mechanism involving rapid binding and inactivation of protein phosphatase catalytic subunit, followed by a slower covalent interaction. It was proposed that inactivation of PP-2A/PP-1 catalytic activity by microcystins precedes covalent adduct formation. In this study, we used a biosensor based on surface plasmon resonance (SPR) to examine the effects of three microcystins, MCLR, MCRR and MCYR, on the binding between PP-2A and its substrate, phosphorylase-a (PL-a), during the first step of the interaction. The SPR biosensor provides real-time information on the association and dissociation kinetics of PL-a with immobilized PP-2A in the absence and presence of microcystins. It was found that the affinity of PL-a to microcystin-bound PP-2A was four times smaller compared to unbound PP-2A, due to 50% decreases in the association rates and two-fold increases in dissociation rates of PL-a binding to PP-2A. The results suggest that the rapid binding of microcystins to the PP-2A catalytic site leads to the formation of a noncovalent microcystin/PP-2A adduct. While the adduct formation fully inhibits the catalytic activity of PP-2A, it only results in partial inhibition of the substrate binding. The similar effects of the three microcystins on PP-2A suggest that the toxins bind to PP-2A at the same site and cause similar conformational changes. The present work also demonstrates the potential application of biosensor technology in environmental toxicological research. (+info)
Glycogen synthase phosphatase interacts with heat shock factor to activate CUP1 gene transcription in Saccharomyces cerevisiae.
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Upon heat shock, transcription of many stress-inducible genes is rapidly and dramatically stimulated by heat shock factor (HSF). A central region of the yeast HSF (designated HSFrr for "repression region") was previously identified and proposed to be involved in repressing the activation domain under non-heat-shock conditions. Here, we used the phage display system to isolate proteins that interact with HSFrr. This should identify factors that modulate HSF activity or directly participate in HSF-mediated transcriptional activation. We constructed a randomly sheared yeast genomic library to express yeast proteins on the surface of lambda phage. HSFrr binding phages were selected by cycles of affinity chromatography. DNA sequencing identified an HSFrr-interacting phage that contains the GAC1 gene. The GAC1 gene encodes the regulatory subunit for a type 1 serine/threonine phosphoprotein phosphatase, Glc7. Both gac1 and glc7 mutations had little effect on HSF activation of gene transcription of two heat shock genes, SSA4 and HSP82. In contrast, heat shock induction of CUP1 gene expression was completely abolished in a glc7 mutant and reduced in a gac1 mutant. The results demonstrate that the Glc7 phosphatase and its Gac1 regulatory subunit play positive roles in HSF activation of CUP1 transcription. (+info)
A cluster of ABA-regulated genes on Arabidopsis thaliana BAC T07M07.
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Arabidopsis thaliana BAC T07M07 encoding the abscisic acid-insensitive 4 (ABI4) locus has been sequenced completely. It contains a 95,713-bp insert and 24 predicted genes. Most putative genes were confirmed by gel-based RNA profiling and a cluster of ABA-regulated genes was identified. One of the 24 genes, designated PP2C5, encodes a putative protein phosphatase 2C. The encoded protein was expressed in Escherichia coli, and its enzyme activity in vitro was confirmed. (+info)
The SV40 large T oncoprotein disrupts DNA-binding of the cell cycle-regulated transcriptional repressor CDF.
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A hallmark of neoplastic transformation by DNA tumor viruses is the deregulation of cell cycle genes. At least in some genes, this deregulation appears to be due to the oncoprotein-mediated disruption of complexes between E2F and pocket proteins and the ensuing generation of transcriptionally active free E2F. In the present study, we have analysed the effect of the SV40 large T oncoprotein (SV-LT) on the function of a different cell cycle-regulated transcriptional repressor, CDF, which is the principal regulator of the cdc25C, cyclin A and cdc2 genes. As shown by genomic footprinting of sorted G1 and G2 cell populations, transformation by SV-LT completely abrogated protection of the CDF binding site (CDE-CHR) in the cdc25C promoter. In agreement with this observation, expression of the SV-LT in fibroblasts led to a dramatic up-regulation of the cdc25C promoter in cells synchronized in G0. These findings indicate that the oncoprotein-mediated dissociation of the CDF repressor protein from its cognate DNA-binding site is a major mechanism in virus-induced transcriptional deregulation. (+info)
The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway.
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Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. We have previously shown that this process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). We now extend our characterization and show that mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. We also find that mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, our results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac. (+info)
RNA splicing: What has phosphorylation got to do with it?
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Many pre-mRNA splicing factors are phosphorylated in vivo, but the role of this modification has been unclear. Recent observations suggest that phosphorylation modulates protein-protein interactions within the spliceosome, thereby contributing to dynamic structural reorganization of the spliceosome during splicing. (+info)