PubMed Health⌕ Search

Biomedical subjects

S Wera

Publications and source records attributed to S Wera.

10 recordsLinked to original sources
PubMed1995

Molecular cloning of NIPP-1, a nuclear inhibitor of protein phosphatase-1, reveals homology with polypeptides involved in RNA processing.

NIPP-1 was originally isolated as a potent and specific nuclear inhibitory polypeptide (16-18 kDa) of protein phosphatase-1. We report here the cDNA cloning of NIPP-1 from bovine thymus and show that the native polypeptide consists of 351 residues and has a calculated mass of 38.5 kDa. The bacterially expressed central third of NIPP-1 completely inhibited the type-1 catalytic subunit, but displayed a reduced inhibitory potency after phosphorylation by protein kinase A and casein kinase 2. Translation of NIPP-1 mRNA in reticulocyte lysates resulted in the accumulation of both intact NIPP-1 and a smaller polypeptide generated by alternative initiation at the codon corresponding to Met143. A data base search showed that the COOH terminus of NIPP-1 is nearly identical to the human ard-1 protein (13 kDa), which has been implicated in RNA processing (Wang, M., and Cohen, S. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Comparison of the cDNAs encoding ard-1 and NIPP-1 suggests that their mRNAs are generated by alternative splicing of the same pre-mRNA. Western blotting with antibodies against the COOH terminus of NIPP-1, however, showed a single polypeptide of 47 kDa, which was enriched in the nucleus. Northern analysis revealed a single transcript of 2.2 kilobases in bovine thymus and of 2.4 kilobases in various human tissues.

Alternative Splicing↗
PubMed1995

Molecular cloning of a human polypeptide related to yeast sds22, a regulator of protein phosphatase-1.

sds22 is a regulatory polypeptide of protein phosphatase-1 that is required for the completion of mitosis in both fission and budding yeast. We report here the cDNA cloning of a human polypeptide that is 46% identical to yeast sds22. The human homolog of sds22 consists of 360 residues, has a calculated molecular mass of 41.6 kDa and shows a tandem array of 11 leucine-rich repeat structures of 22 residues. Northern analysis revealed a major transcript of 1.39 kb in all 8 investigated human tissues. sds22 was detected by western analysis in both the cytoplasm and the nucleus of rat liver cells as a polypeptide of 44 kDa.

Amino Acid Sequence↗
PubMed1995

Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells.

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M. (1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.

Animals↗
PubMed1995

Rapamycin, FK506 and cyclosporin A inhibit human prolactin gene expression.

In this work we demonstrate that transcription of the human prolactin gene is inhibited by the immunosuppressants FK506 (IC50 = 25 nM), cyclosporin A (IC50 = 190 nM) and rapamycin (IC50 = 25 nM). Whereas the effect of FK506 and cyclosporin A is specific for prolactin gene transcription, rapamycin has a more general effect on transcription and/or translation in pituitary cells. In view of recent work demonstrating the immunoactivating role of prolactin, these results suggest that inhibition of prolactin gene expression in the pituitary may contribute to the mechanism of action of immunosuppressants.

Animals↗
PubMed1995

Cyclosporin A, rapamycin and FK506 decrease prolactin release from rat pituitary cells in primary culture.

It is at present well established that prolactin exerts a non-specific immunoactivating function. In this work we tested whether the immunosuppressant drugs cyclosporin A, FK506 and rapamycin influence prolactin release from rat pituitary cells in primary culture. The tested drugs had no effect on the prolactin release measured during a 2h incubation period, indicating that they do not influence the secretion of prolactin from intracellular stores into the culture medium. During longer incubation times (48h), however, prolactin release was diminished to 56% +/- 18 (10 microM cyclosporin A), 64% +/- 14 (1 microM rapamycin) or 64% +/- 7 (1 microM FK506), suggesting an effect on prolactin production. At these drug concentrations no toxic effects were observed. The data indicate that inhibition of pituitary prolactin synthesis might contribute to the immunosuppressant action of cyclosporin A, rapamycin and FK506.

Animals↗
PubMed1994

Calcineurin as a possible new target for treatment of Parkinson's disease.

It is hypothesized that the immunosuppressive agents cyclosporin A and FK-506 may elicit a dopamine-like effect upon dopaminoceptive neurons in the striatum. When complexed to their immunophilins, these molecules will inhibit calcineurin activity leading to increased phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and hence, inhibition of protein phosphatase-1 activity. As a net result, intracellular protein phosphorylation increases. One or more of these proteins may, in their phosphorylated form, inhibit the depolarization of the neurons, resulting in a dopamine-like effect.

Calcineurin↗
PubMed1993

Okadaic acid, a protein phosphatase inhibitor, enhances transcription of a receptor gene containing sequence A of the human prolactin promoter.

Human PRL (hPRL) gene expression is controlled by cAMP and Ca2+. This control is mediated by two cis-elements: a Pit-1 binding site (-62 to -35) and sequence A (-110 to -85), present in the hPRL promoter. We have investigated whether protein phosphatases could be involved in this regulation. GC-type rat pituitary tumor cells were transfected with sequence -138 to -35 of the hPRL gene promoter, upstream from a thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Addition of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, stimulates transient expression of the CAT gene. The dose-response curve shows a maximal effect at 25 nM OA (2.2-fold stimulation above controls). The OA effect is also observed with a natural 4500-base pair hPRL promoter. A single copy of the hPRL promoter sequence -115 to -85 (sequence A) confers to a thymidine kinase-CAT construct an identical response to OA, whereas a single copy of the proximal Pit-1 binding site does not. Synergism is observed between cAMP and OA in activating PRL gene transcription. This synergism is also observed with a single copy of sequence A. The effect of cAMP is not mediated by an L-type Ca2+ channel, since addition of the Ca2+ channel antagonist verapamil does not decrease it, nor does complexing extracellular Ca2+ significantly reduce it. Furthermore, OA and the Ca2+ channel opener BAY K8644 exert additive effects.

Calcium↗
PubMed1991

Purification and characterization of the glycogen-bound protein phosphatase from rat liver.

Glycogen-bound protein phosphatase G from rat liver was transferred from glycogen to beta-cyclodextrin (cycloheptaamylose) linked to Sepharose 6B. After removal of the catalytic subunit and of contaminating proteins with 2 M NaCl, elution with beta-cyclodextrin yielded a single protein on native polyacrylamide gel electrophoresis and two polypeptides (161 and 54 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several lines of evidence indicate that the latter polypeptides are subunits of the protein phosphatase G holoenzyme. First, these polypeptides were also present, together with the catalytic subunit, in the extensively purified holoenzyme. Also, polyclonal antibodies against these polypeptides were able to bind the holoenzyme. Further, while bound to cyclodextrin-Sepharose, the polypeptides were able to recombine with separately purified type-1 (AMD) catalytic subunit, but not with type-2A (PCS) catalytic subunit. The characteristics of the reconstituted enzyme resembled those of the nonpurified protein phosphatase G. At low dilutions, the spontaneous phosphorylase phosphatase activity of the reconstituted enzyme was about 10 times lower than that of the catalytic subunit, but it was about 1000-fold more resistant to inhibition by the modulator protein (inhibitor-2). In contrast with the free catalytic subunit, the reconstituted enzyme co-sedimented with glycogen, and it was able to activate purified liver glycogen synthase b. Also, the synthase phosphatase activity was synergistically increased by a cytosolic phosphatase and inhibited by physiological concentrations of phosphorylase alpha and of Ca2+.

Animals↗