Purification and Characterization of Two Potent Heat-Stable Protein Inhibitors of Protein Phosphatase 2A from Bovine Kidney

Li, Mei; Guo, Hong; Damuni, Zahi

doi:10.1021/bi00006a020

Cited by 211 publications

(245 citation statements)

References 34 publications

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“…Such an inhibition level is the same as that contributed by a specific PP-2A inhibitor, PP2A-I1 [65], suggesting that 10 nM okadaic acid can inhibit PP-2A. This is supported by the finding that treatment of cell extracts with a mixture of PP-2A inhibitor and 10 nM okadaic acid gives the same inhibition as each individual component does.…”

Section: Discussionmentioning

confidence: 65%

“…To examine why 10 nM okadaic acid causes a 20% decrease in total phosphatase activity, we used a specific PP-2A inhibitor, PP2A-I1 [65]. When this inhibitor was used at a concentration three times higher than the IC 50 [65] in our phosphatase activity assays, approximately 15% of the total phosphatase activity was inhibited, an amount very close to that blocked by 10 nM okadaic acid ( Table 1 and Fig. 2A); this suggests that 10 nM okadaic acid is able to inhibit PP-2A.…”

Section: Resultsmentioning

confidence: 99%

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Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Fass

Huizar

et al. 1998

European Journal of Biochemistry

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It is well established that phosphorylation and dephosphorylation are key cellular events which regulate important metabolic activities such as gene expression, cell cycle progression, and apoptosis. The polyether fatty acid, okadaic acid has been shown previously to activate apoptosis in a variety of cell lines. Although this marine sponge toxin is known to inhibit protein phosphatase (PP)-2A and PP-1, it is not certain in most cases whether inhibition of PP-1 or PP-2A is necessary to activate apoptosis. Furthermore, it is not clear how inhibition of these phosphatases leads to apoptosis. Here we present evidence that inhibition of PP-2A by okadaic acid does not activate apoptosis in the lens system. However, when PP-1 is inhibited by okadaic acid, rabbit lens epithelial cells undergo rapid apoptosis. Associated with this process is the several-fold up-regulation of the tumor suppressor gene p53 and the pro-apoptotic gene bax at both mRNA and protein levels. Analyses of the temporal pattern of expression of the two genes reveal that the up-regulation is maximized in a few hours after treatment with okadaic acid, when the majority of the treated cells become committed to apoptosis. A brief treatment of the cells with a protein synthesis inhibitor can abolish okadaic acid-induced up-regulation of both P53 and Bax proteins. Concomitant with this inhibition, okadaic acid-induced apoptosis is also temporarily blocked. These results suggest that okadaic acid-induced expression of p53, bax, and other genes are necessary for the activation of the apoptotic programs in lens systems.Keywords : apoptosis; p53 ; bax ; phosphatase-1; lens.Apoptosis is a distinct form of cell death regulated by internal genetic programs [1,2]. The morphological changes associated with apoptosis include blebbing of cytoplasmic membranes, condensation of nucleoplasm and cytoplasm, and fragmentation of the dying cell into apoptotic bodies which are phagocytosed by neighboring cells [3,4]. The biochemical hallmarks of apoptosis are marked by DNA fragmentation into nucleosomal fragments [4,5], activation of the interleukin-1β-converting enzyme family of proteases [6], and cleavage of various substrates of that family of proteases such as poly(ADPribose) polymerase [7,8] and nuclear lamin [9].Apoptosis normally occurs as a physiological process during certain stages of animal development or during tissue homeostasis in an adult organism [10,11]. It can also be triggered by various external signals such as hormone [4], γ-irradiation [12] and withdrawal of growth factors [13] and other factors (reviewed in [2]). Normal physiological apoptosis helps to remove

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Section: Discussionmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Fass

Huizar

et al. 1998

European Journal of Biochemistry

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“…PHAPI is also known as pp32, a potent tumor suppressor (12-15) that is down-regulated in cancer with reciprocal increased expression of pp32r1 and pp32r2, other members of the pp32 gene family (12). Although the physiological role of PHAPI/pp32 is not fully understood, it has been shown to be a potent inhibitor of PP2A in vitro (16,17) and also promotes apoptosis by caspase-9 activation (18).In previous studies, we found that dietary lectins that recognize the oncofetal Thomsen-Friedenreich (TF) carbohydrate antigen (galactose ␤1-3 N-acetylgalactosamine ␣-) can cause marked effects on the growth of human intestinal epithelial cancer cells (19 -23). Since TF antigen can also be a ligand for mammalian lectins including members of the galectin family (24, 25), we have undertaken further investigation of the mechanism underlying these effects.…”

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confidence: 99%

Protein Phosphatase 2A, a Negative Regulator of the ERK Signaling Pathway, Is Activated by Tyrosine Phosphorylation of Putative HLA Class II-associated Protein I (PHAPI)/pp32 in Response to the Antiproliferative Lectin, Jacalin

Yu¹,

Packman

Weldon

et al. 2004

Journal of Biological Chemistry

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Protein phosphatase 2A (PP2A) is a family of mammalian serine/threonine phosphatases that is involved in the control of many cellular functions including those mediated by extracellular signal-regulated kinase (ERK) signaling. While investigating the reversible antiproliferative effect of the dietary lectin, jacalin, which binds the Thomsen-Friedenreich antigen (galactose ␤1-3 N-acetylgalactosamine ␣-), we have found that this lectin (30 g/ml) induces rapid, transient, tyrosine phosphorylation of putative human HLA-DR-associated protein I (PHAPI, also known as the tumor suppressor pp32) in HT29 human colon cancer cells. This is accompanied by the release of PP2A from association with PHAPI, allowing increased phosphatase activity of PP2A (by 42 ؎ 10% at 10 min) and consequent complete dephosphorylation of the ERK kinase, MEK1/2, by 10 min and of ERK1/2 by 60 min. PHAPI knockdown by RNA interference abolished the effects of jacalin on PP2A activation and MEK inhibition. Thus phosphorylation of PHAPI/pp32 is a critical regulatory step in PP2A activation and ERK signaling.Protein phosphatase 2A (PP2A) 1 is a family of mammalian serine/threonine phosphatases that accounts for the major portion of serine/threonine kinase activity in most cells. PP2A is a trimeric holoenzyme complex and is composed of a catalytic C subunit, a structural A subunit, and a regulatory B-type subunit, each encoded by separate genes (1, 2). Up to 75 different trimeric ABC holoenzymes may exist. The C subunit is the enzymatically active component, A is a scaffolding component, and B acts as a targeting module that directs the enzyme to various intracellular locations and also provides substrate specificity.PP2A was initially thought to act passively and nonspecifically in antagonizing the action of kinases by removal of phosphate groups from their substrates, but it has become clear that it is tightly regulated by mechanisms that include variation in its subunit composition (3), phosphorylation, and/or methylation of its catalytic subunit and signal-regulated targeting to specific subcellular compartments (1-4). PP2A is an important negative regulator of the extracellular signal-regulated kinase (ERK) signaling pathway (5-7) and is involved in the control of many cellular functions including metabolism, transcription, translation, RNA splicing, DNA replication, cell cycle progression, transformation, and apoptosis (1-8).PHAPI, first purified by affinity with an agarose-conjugated synthetic cytoplasmic region of HLA-DR ␣ chain, is a putative HLA class II-associated cytosolic protein (9), later identified as a phosphoprotein (10). It belongs to a group of proteins containing 20 -29-residue leucine-rich repeat motifs. The principal function of these motifs seems to be the provision of a structural framework that allows protein-protein interactions (11). PHAPI is also known as pp32, a potent tumor suppressor (12-15) that is down-regulated in cancer with reciprocal increased expression of pp32r1 and pp32r2, other members of the pp32 gene family (...

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“…and I 2 PP2A [13,14] . In vitro, the PP2A activity is dramatically inhibited after tyrosine 307 phosphorylation [24,25] .…”

Section: Pp2amentioning

confidence: 99%

“…Holoenzyme composition and post-translational modification have been suggested to modulate PP2A activity. Besides, some intracellular small inhibitory molecules such as I 1 PP2A and I 2 PP2A may bind to and directly inhibit PP2A [13,14] .…”

Section: Introductionmentioning

confidence: 99%

Zinc binds to and directly inhibits protein phosphatase 2A in vitro

et al. 2015

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Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn 2+ has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn 2+ on PP2A and their direct interaction in vitro. The results showed that Zn 2+ mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 μmol/L) of Zn 2+ inhibited PP2A directly. Further Zn 2+ -IDA-agarose affinity binding assays showed that Zn 2+ bound to and inhibited PP2Ac but not PP2Ac or PP2Ac . Taken together, Zn 2+ inhibits PP2A directly through binding to PP2Ac in vitro.

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Purification and Characterization of Two Potent Heat-Stable Protein Inhibitors of Protein Phosphatase 2A from Bovine Kidney

Cited by 211 publications

References 34 publications

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Protein Phosphatase 2A, a Negative Regulator of the ERK Signaling Pathway, Is Activated by Tyrosine Phosphorylation of Putative HLA Class II-associated Protein I (PHAPI)/pp32 in Response to the Antiproliferative Lectin, Jacalin

Zinc binds to and directly inhibits protein phosphatase 2A in vitro

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