Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

pRb controls cell proliferation by restricting inappropriate entry of cells into the cell division cycle. As dephosphorylation of pRb during mitotic exit activates its growth suppressive function, identification of the protein phosphatase that dephosphorylates pRb, and characterization of the mechanism of its regulation, are essential to elucidating the mechanisms of cell growth control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing analyses indicate that the catalytic enzyme (PP1c) is present in a high molecular weight complex, with a predicted molecular mass of 166 kDa. PP1-interacting proteins in the mitotic cell extracts are identified. Two PP1-interacting proteins (41 and 110 kDa) are shown to form distinct complexes with PP1c from fractions of separated mitotic cell extracts containing phosphorylase phosphatase activity. However, only the 110-kDa PP1-interacting protein is present in fractions containing pRb-directed phosphatase activity, identifying this protein as a putative activator of PP1 function toward pRb during mitosis.
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PMID:High molecular weight protein phosphatase type 1 dephosphorylates the retinoblastoma protein. 902 Jan 79

We have previously reported on the M-phase specific dephosphorylation of pRb and identified a type 1 serine/threonine protein phosphatase (PP1) as the enzyme mediating pRb dephosphorylation. In this report, we have characterized the pRb-directed phosphatase activity found in mitotic cells with respect to dose dependence and demonstrate that the pRb isoform conversion detected in vitro mirrors the pRb isoform conversion which occurs during mitosis of intact cells. Cell fractionation and PP1 catalytic subunit isolation studies support the notion that the pRb-directed phosphatase activity involves subpopulations of PP1 catalytic subunits. Coprecipitation studies revealed that PP1 can form a complex with hypophosphorylated pRb which was converted from the hyperphosphorylated form in mitotic cell extracts. Taken together with data from previous reports in the literature, a model for the regulation of PP1 activity towards pRb during mitotic exit is proposed.
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PMID:Characterization of the mitotic phase pRb-directed protein phosphatase activity. 918 55

The retinoblastoma gene product (pRb) is dephosphorylated at the exit from mitosis and protein phosphatase-1 (PP1) seems to be responsible for such dephosphorylation. Three isoforms of PP1 exist in mammalian cells, alpha, gamma1 and delta, with differential subcellular localization and potentially different targeting subunits and functions. In order to identify which isoform dephosphorylates pRb, we used isoform-specific antibodies and analyzed the association of the PP1 isoforms with pRb in nocodazole-blocked (mitotic) HeLa cells and in cells released from the mitotic block (early G1). PP1delta was found associated with the pRb immunoprecipitated from a mitotic cell extract, whereas neither PP1gamma1 nor PP1alpha were detected. In G1 cells progressively less pRb and of lower Mr was detected in anti-PP1delta immunocomplexes, and pRb had almost disappeared by 8 h. The PP1 associated with pRb was inactive at mitosis, but underwent a quick activation as cells exited from mitosis, with a peak at 1 h. Then the activity decreased progressively and disappeared by 8 h. [32P]labeled pRb, obtained from G2 cells, was dephosphorylated "in vitro" by PP1delta obtained from early G1 cells. Altogether, the results indicated that PP1delta associated with pRb and may be responsible for the phosphatase activity detected in the pRb complexes, supporting the hypothesis that PP1delta may be the isoform that dephosphorylates pRb.
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PMID:Association of protein phosphatase-1delta with the retinoblastoma protein and reversible phosphatase activation in mitotic HeLa cells and in cells released from mitosis. 920 24

The tumour suppressor PTEN, also named MMAC1 or TEP1, is associated with a number of malignancies in human populations. This protein has a dual protein phosphatase activity, being also capable to dephosphorylate phosphatidylinositol 3,4,5 triphosphate. We have studied the mechanism of growth suppression attributable to PTEN. We observed that PTEN overexpression inhibits cell growth in a variety of normal and transformed, human and murine cells. Bromodeoxyuridine (BrdU) incorporation and TUNEL labelling experiments in transiently transfected cells demonstrate that this inhibition is due to a cell cycle arrest rather than induction of apoptosis. Given that PTEN is unable to cause cell growth arrest in retinoblastoma (Rb)-deficient cell lines, we have explored the possible requirement for pRb in the PTEN-induced inhibition of cell proliferation. We found that the co-expression of SV40 antigen, but not a mutant form (which binds exclusively to p53), and cyclin D1/cdk4 are able to overcome the PTEN-mediated growth suppression. In addition, the reintroduction of a functional pRb, but not its relatives p107 or p130, in Rb-deficient cells restores the sensitivity to PTEN-induced arrest. Finally, the hyperphosphorylation of transfected pRb is inhibited by PTEN co-expression and restored by PI-3K co-expression. Accordingly, PTEN gene is mostly expressed, in parallel to Akt, in mid-late G1 phase during cell cycle progression prior to pRb hyperphosphorylation. Finally, we have studied the signal transduction pathways modulated by PTEN expression. We found that PTEN-induced growth arrest can be rescued by the co-expression of active PI-3K and downstream effectors such as Akt or PDK1, and also certain small GTPases such as Rac1 and Cdc42, but not by active Ha-ras, raf or RhoA. Collectively, our data link the tumour suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signalling molecules whose final target is the functional inactivation of the retinoblastoma gene product.
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PMID:PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. 1060 5

Ceramide is known to induce pRb (retinoblastoma gene product) dephosphorylation through the activation of ceramide-activated protein phosphatase (CAPP) during G1 arrest, but other molecular mechanisms linked to regulation of pRb dephosphorylation during ceramide-induced G1 arrest are poorly understood. In this paper, we investigated whether p21, a cdk (cyclin-dependent kinase) inhibitor, is involved in the induction of pRb dephosphorylation during ceramide-induced G1 arrest. In SK-Hep-1 cells, the addition of ceramide resulted in pRb dephosphorylation and G1 arrest. The activity of cdk2 was inhibited in response to ceramide during this process. p21 protein and mRNA were remarkably induced, while the protein level of p53, known as a transcriptional activator of p21, was not elevated at the same condition. p21 induction was also observed in the Hep3B cells lacking a functional p53 after exposure to ceramide. Although p21 is induced in ceramide-treated Hep3B cells, Hep3B cells do not induce G1 arrest, because Hep3B cells are deficient in a functional pRb protein. To confirm that pRb is a critical target for the induction of G1 arrest by inhibiting cdk2 activity through p53-independent p21, pRb-expressing vector was transfected into Hep3B cells. After treatment with ceramide, pRb-expressing cells (pRb+/+), but not pRb-/- cells, were arrested in G1 phase. In pRb+/+ cells, ceramide-mediated G1 arrest was accompanied by the accumulation of hypophosphorylated pRb and p21 associated with cdk2. Together, these results suggest that p21, induced through p53-independent pathway, participates in the induction of pRb dephosphorylation by inhibiting cdk2 activity during ceramide-mediated G1 arrest in hepatocarcinoma cells.
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PMID:Induction of p53-independent p21 during ceramide-induced G1 arrest in human hepatocarcinoma cells. 1087 74

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2.
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PMID:Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130. 1262 Oct 62

The Rb protein suppresses development of an abnormal state of endoreduplication arising after S phase DNA damage. In diploid, S phase cells, the activity of protein phosphatase 2A (PP2A) licenses the stable association of un(der)phosphorylated Rb with chromatin. After damage, chromatin-associated pRb is attracted to certain chromosomal replication initiation sites in the order in which they normally fire. Like S phase DNA damage in Rb(-/-) cells, specific interruption of PP2A function in irradiated, S phase wt cells also elicited a state of endoreduplication. Thus, PP2A normally licenses the recruitment of Rb to chromatin sites in S phase from which, after DNA damage, it relocalizes to selected replication control sites and suppresses abnormal, postdamage rereplicative activity.
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PMID:Active localization of the retinoblastoma protein in chromatin and its response to S phase DNA damage. 1452 18

The functions of the retinoblastoma protein (pRb) are in part regulated by reversible and cell cycle-dependent phosphorylation. While the regulation of pRb by cyclin-dependent kinases (Cdks) has been studied extensively, the role(s) of protein phosphatase 1 (PP1) in controlling pRb are only partially understood. In this chapter, we will describe experimental approaches to investigate the interactions between pRb and PP1. Methods will be presented to study the cell cycle-dependent dephosphorylation of pRb by various PP1 isozymes, the specificity of PP1 isozymes for distinct pRb phosphorylation sites, the dephosphorylation of pRb associated with apoptosis, and the cell cycle- and pRb-dependent phosphorylation of PP1.
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PMID:Interaction between the retinoblastoma protein and protein phosphatase 1 during the cell cycle. 1522 May 19

In sympathetic neurons, C6-ceramide, as well as endogenous ceramides, blocks apoptosis elicited by NGF (nerve growth factor) deprivation. The mechanism(s) involved in ceramide-induced neuronal survival are poorly understood. Few direct targets for the diverse cellular effects of ceramide have been identified. Amongst those proposed is PP-1c, the catalytic subunit of serine/threonine PP-1 (protein phosphatase-1). Here, we present the first evidence of PP-1c activation by ceramide in live cells, namely NGF-deprived sympathetic neurons. We first determined PP activity in cellular lysates from sympathetic neurons treated with exogenous ceramide and demonstrated a 2-3-fold increase in PP activity. PP activation was completely blocked by the addition of the specific type-1 PP inhibitor protein I-2 as well as by tautomycin, but unaffected by 2 nM okadaic acid, strongly indicating that the ceramide-activated phosphatase activity was PP-1c. Inhibition of PP activity by phosphatidic acid (which has been reported to be a selective inhibitor of PP-1c) and tautomycin (a PP-1 and PP-2A inhibitor), but not by 10 nM okadaic acid, abolished the anti-apoptotic effect of ceramide in NGF-deprived neurons, suggesting that activation of PP-1c is required for ceramide-induced neuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1c activation, suggesting that two consequences of NGF deprivation in sympathetic neurons are inhibition of PP-1c and subsequent hyperphosphorylation of pRb protein. These findings suggest a novel mechanism for ceramide-induced survival, and implicate the involvement of PPs in apoptosis induced by NGF deprivation.
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PMID:Activation of serine/threonine protein phosphatase-1 is required for ceramide-induced survival of sympathetic neurons. 1536 Oct 69

Based on previous studies, a minimal set of genetic alterations that is required to convert normal human fibroblasts into cancer cells has been defined. Essential roles for telomere maintenance and alterations in phosphatase 2A activity were inferred from experiments in which tumorigenicity was tested by injecting cells under the skin of immunodeficient mice. However, in the present experiments, the combination of SV40 large T antigen and activated Ras, without hTERT or SV40 small t antigen, was sufficient to convert nine different primary human fibroblast cell strains to a fully malignant state. The malignant behavior of the cells was demonstrated by growth of the cells into invasive tumors when the cells were injected beneath the kidney capsule of immunodeficient mice. Lung metastases and circulating tumor cells were also detected. These tumors were not immortal; cells entered crisis, from which they could be rescued by expression of hTERT. However, the same cell populations were not tumorigenic when they were injected under the skin. In this site, tumorigenicity required the expression of hTERT and SV40 small t antigen as well as SV40 large T antigen and Ras. The cellular pathways targeted by SV40 large T antigen (p53 and pRb) and those targeted by activated Ras represent a minimal set of genetic alterations required for the conversion of normal human fibroblasts into cancer cells.
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PMID:The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay. 1603 9


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