Cell Cycle Dependent Association of EBP50 with Protein Phosphatase 2A in Endothelial Cells

Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport.

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Troglitazone acutely inhibits protein synthesis in endothelial cells via a novel mechanism involving protein phosphatase 2A-dependent p70 S6 kinase inhibition

AJP Cell Physiology ◽

10.1152/ajpcell.00491.2005 ◽

2006 ◽

Vol 291 (2) ◽

pp. C317-C326 ◽

Cited By ~ 36

Author(s):

Du-Hyong Cho ◽

Yoon Jung Choi ◽

Sangmee Ahn Jo ◽

Jungsang Ryou ◽

Jin Yi Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Synthesis ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Peroxisome Proliferator ◽

Inhibitory Effects ◽

S6 Kinase ◽

P70 S6 Kinase ◽

Inhibition Of Protein Synthesis ◽

Phosphatase 2A

Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligands, have been implicated in the inhibition of protein synthesis in a variety of cells, but the underlying mechanisms remain obscure. We report that troglitazone, the first TZD drug, acutely inhibited protein synthesis by decreasing p70 S6 kinase (p70S6K) activity in bovine aortic endothelial cells (BAEC). This inhibition was not accompanied by decreased phosphorylation status or in vitro kinase activity of mammalian target of rapamycin (mTOR). Furthermore, cotreatment with rapamycin, a specific mTOR inhibitor, and troglitazone additively inhibited both p70S6K activity and protein synthesis, suggesting that the inhibitory effects of troglitazone are not mediated by mTOR. Overexpression of the wild-type p70S6K gene significantly reversed the troglitazone-induced inhibition of protein synthesis, indicating an important role of p70S6K. Okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, partially reversed the troglitazone-induced inhibition of p70S6K activity and protein synthesis. Although troglitazone did not alter total cellular PP2A activity, it increased the physical association between p70S6K and PP2A, suggesting an underlying molecular mechanism. GW9662, a PPARγ antagonist, did not alter any of the observed inhibitory effects. Finally, we also found that the mTOR-independent inhibitory mechanism of troglitazone holds for the TZDs ciglitazone, pioglitazone, and rosiglitazone, in BAEC and other types of endothelial cells tested. In conclusion, our data demonstrate for the first time that troglitazone (and perhaps other TZDs) acutely decreases p70S6K activity through a PP2A-dependent mechanism that is independent of mTOR and PPARγ, leading to the inhibition of protein synthesis in endothelial cells.

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Characterization of the Aalpha and Abeta subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution

Biochemical Journal ◽

10.1042/bj20021244 ◽

2003 ◽

Vol 369 (2) ◽

pp. 387-398 ◽

Cited By ~ 59

Author(s):

Jin ZHOU ◽

Huong T. PHAM ◽

Ralf RUEDIGER ◽

Gernot WALTER

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Expression Patterns ◽

Simian Virus 40 ◽

Polyoma Virus ◽

Tumour Antigen ◽

Small Tumour ◽

Phosphatase 2A

Protein phosphatase 2A (PP2A) is very versatile owing to a large number of regulatory subunits and its ability to interact with numerous other proteins. The regulatory A subunit exists as two closely related isoforms designated Aα and Aβ. Mutations have been found in both isoforms in a variety of human cancers. Although Aα has been intensely studied, little is known about Aβ. We generated Aβ-specific antibodies and determined the cell cycle expression, subcellular distribution, and metabolic stability of Aβ in comparison with Aα. Both forms were expressed at constant levels throughout the cell cycle, but Aα was expressed at a much higher level than Aβ. Both forms were found predominantly in the cytoplasm, and both had a half-life of approx. 10h. However, Aα and Aβ differed substantially in their expression patterns in normal tissues and in tumour cell lines. Whereas Aα was expressed at similarly high levels in all tissues and cell lines, Aβ expression varied greatly. In addition, in vivo studies with epitope-tagged Aα and Aβ subunits demonstrated that Aβ is a markedly weaker binder of regulatory B and catalytic C subunits than Aα. Construction of phylogenetic trees revealed that the conservation of Aα during the evolution of mammals is extraordinarily high in comparison with both Aβ and cytochrome c, suggesting that Aα is involved in more protein—protein interactions than Aβ. We also measured the binding of polyoma virus middle tumour antigen and simian virus 40 (SV40) small tumour antigen to Aα and Aβ. Whereas both isoforms bound polyoma virus middle tumour antigen equally well, only Aα bound SV40 small tumour antigen.

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The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle 1 1Edited by J. Karn

Journal of Molecular Biology ◽

10.1006/jmbi.2000.4062 ◽

2000 ◽

Vol 302 (1) ◽

pp. 103-119 ◽

Cited By ~ 20

Author(s):

Christine Van Hoof ◽

Veerle Janssens ◽

Ivo De Baere ◽

Johannes H de Winde ◽

Joris Winderickx ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Yeast Cell ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Yeast Cell Cycle ◽

G1 Phase ◽

Phosphatase 2A

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Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei

Molecular and Biochemical Parasitology ◽

10.1016/j.molbiopara.2014.04.006 ◽

2014 ◽

Vol 194 (1-2) ◽

pp. 48-52 ◽

Cited By ~ 2

Author(s):

Karen G. Rothberg ◽

Neal Jetton ◽

James G. Hubbard ◽

Daniel A. Powell ◽

Vidya Pandarinath ◽

...

Keyword(s):

Cell Cycle ◽

Family Member ◽

Trypanosoma Brucei ◽

Protein Phosphatase ◽

Cell Cycle Progression ◽

Protein Phosphatase 2A ◽

Cycle Progression ◽

Phosphatase 2A

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Interrelationship between protein phosphatase-2A and cytoskeletal architecture during the endothelial cell response to soluble products produced by human head and neck cancer

Otolaryngology ◽

10.1067/mhn.2000.105186 ◽

2000 ◽

Vol 122 (5) ◽

pp. 721-727 ◽

Cited By ~ 1

Author(s):

Christopher J. Witt ◽

Steven P. Gabel ◽

Jeremy Meisinger ◽

Gwendolyn Werra ◽

Shirley W. Liu ◽

...

Keyword(s):

Endothelial Cells ◽

Retinoic Acid ◽

Endothelial Cell ◽

Cell Motility ◽

Head And Neck ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Human Head ◽

Progressive Development ◽

Phosphatase 2A

Tumor neovascularization is necessary for the progressive development of all solid tumors, including head and neck squamous cell carcinomas (HNSCCs). The angiogenic process includes increased endothelial cell motility. Our prior studies have shown the importance of protein phosphatase-2A (PP-2A) in restricting endothelial cell motility. Because motility is regulated by the polymerization/depolymerization of the cellular cytoskeleton, the present study defined the interrelationship between PP-2A and the cytoskeleton during endothelial cell responses to HNSCC-derived angiogenic factors. PP-2A was shown to colocalize with microtubules of unstimulated endothelial cells. However, exposure to HNSCC-derived products resulted in a more diffuse distribution of PP-2A staining and a loss of filamentous tubulin. The feasibility of pharmacologically preventing this cytoskeletal disorganization as a means of blocking tumor-induced angiogenesis was tested. This was accomplished by use of 1α,25-dihydroxyvitamin D3 [1,25 (OH)2D3] and all- trans-retinoic acid to indirectly stimulate PP-2A activity through their capacity to elevated intracellular levels of the second messenger ceramide. Pretreatment of endothelial cells with either 1,25(OH)2D3 or retinoic acid prevented the cytoskeletal disorganization that otherwise occurs in endothelial cells on exposure to HNSCC-derived products. These studies support the feasibility of using elevation of PP-2A to prevent the morphogenic component of the angiogenic process that is stimulated by HNSCC-derived factors.

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Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3242 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3242-3253 ◽

Cited By ~ 68

Author(s):

Y Shu ◽

H Yang ◽

E Hallberg ◽

R Hallberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

High Temperatures ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Molecular Genetic Analysis ◽

Regulatory Subunit ◽

Temperature Sensitive ◽

Checkpoint Control ◽

Phosphatase 2A

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.

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The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.e10-06-0487 ◽

2011 ◽

Vol 22 (1) ◽

pp. 20-32 ◽

Cited By ~ 25

Author(s):

Sidonie Wicky ◽

Hendri Tjandra ◽

David Schieltz ◽

John Yates ◽

Douglas R. Kellogg

Keyword(s):

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Regulatory Subunit ◽

Cyclin Dependent Kinase ◽

Cdc25 Phosphatase ◽

Phosphatase 2A ◽

Wee1 Kinase ◽

Cycle Dependent ◽

Related Proteins ◽

Stoichiometric Complex

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2ACdc55). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2ACdc55and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle–dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2ACdc55and suggest that upstream signals that regulate PP2ACdc55may play an important role in controlling entry into mitosis.

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