scholarly journals A Novel Assay for Protein Phosphatase 2A (PP2A) Complexes In Vivo Reveals Differential Effects of Covalent Modifications on Different Saccharomyces cerevisiae PP2A Heterotrimers

2005 ◽  
Vol 4 (6) ◽  
pp. 1029-1040 ◽  
Author(s):  
Matthew S. Gentry ◽  
Yikun Li ◽  
Huijun Wei ◽  
Farhana F. Syed ◽  
Sameer H. Patel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Posttranslational Modifications ◽  
Protein Phosphatase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A ◽  
Covalent Modifications

ABSTRACT Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.

scholarly journals The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo.

1994 ◽  
Vol 269 (23) ◽  
pp. 16311-16317 ◽  
Author(s):  
B. Favre ◽  
S. Zolnierowicz ◽  
P. Turowski ◽  
B.A. Hemmings
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A

scholarly journals Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A

2014 ◽  
Vol 395 (7-8) ◽  
pp. 881-889 ◽  
Author(s):  
Christian Löw ◽  
Esben M. Quistgaard ◽  
Michael Kovermann ◽  
Madhanagopal Anandapadamanaban ◽  
Jochen Balbach ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Posttranslational Modifications ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Structural Basis ◽  
Interaction Site ◽  
Interaction Partners ◽  
Phosphatase 2A ◽  
Additional Interaction

Abstract Protein phosphatase 2A (PP2A) is a highly abundant heterotrimeric Ser/Thr phosphatase involved in the regulation of a variety of signaling pathways. The PP2A phosphatase activator (PTPA) is an ATP-dependent activation chaperone, which plays a key role in the biogenesis of active PP2A. The C-terminal tail of the catalytic subunit of PP2A is highly conserved and can undergo a number of posttranslational modifications that serve to regulate the function of PP2A. Here we have studied structurally the interaction of PTPA with the conserved C-terminal tail of the catalytic subunit carrying different posttranslational modifications. We have identified an additional interaction site for the invariant C-terminal tail of the catalytic subunit on PTPA, which can be modulated via posttranslational modifications. We show that phosphorylation of Tyr307PP2A-C or carboxymethylation of Leu309PP2A-C abrogates or diminishes binding of the C-terminal tail, whereas phosphorylation of Thr304PP2A-C is of no consequence. We suggest that the invariant C-terminal residues of the catalytic subunit can act as affinity enhancer for different PP2A interaction partners, including PTPA, and a different ‘code’ of posttranslational modifications can favour interactions to one subunit over others.

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling

2001 ◽  
Vol 353 (3) ◽  
pp. 417-439 ◽  
Author(s):  
Veerle JANSSENS ◽  
Jozef GORIS
Keyword(s):  
Protein Phosphatase ◽  
Cellular Localization ◽  
Protein Phosphatase 2A ◽  
Viral Proteins ◽  
Catalytic Subunit ◽  
Post Translational Modification ◽  
Regulatory Subunits ◽  
Regulatory Ability ◽  
Phosphatase 2A ◽  
Cycle Regulation

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

scholarly journals Localization of Saccharomyces cerevisiae Protein Phosphatase 2A Subunits throughout Mitotic Cell Cycle

2002 ◽  
Vol 13 (10) ◽  
pp. 3477-3492 ◽  
Author(s):  
Matthew S. Gentry ◽  
Richard L. Hallberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Phosphatase ◽  
Fluorescent Protein ◽  
Protein Phosphatase 2A ◽  
Mitotic Cell ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
C Subunit ◽  
Phosphatase 2A

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit.

scholarly journals Adenovirus Protein E4orf4 Induces Premature APCCdc20 Activation in Saccharomyces cerevisiae by a Protein Phosphatase 2A-Dependent Mechanism

2010 ◽  
Vol 84 (9) ◽  
pp. 4798-4809 ◽  
Author(s):  
Melissa Z. Mui ◽  
Diana E. Roopchand ◽  
Matthew S. Gentry ◽  
Richard L. Hallberg ◽  
Jackie Vogel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Functional Characterization ◽  
Specific Inhibitor ◽  
Human Adenovirus ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Phosphatase 2A

ABSTRACT Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G2/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2ACdc55 in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2ACdc55 in regulating APCCdc20 activity.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

Structure and transcriptional regulation of protein phosphatase 2A catalytic subunit genes [Erratum to document cited in CA114(3):18572v]

Biochemistry ◽  
1991 ◽  
Vol 30 (21) ◽  
pp. 5328-5328 ◽  
Author(s):  
Yeesim Khew-Goodall ◽  
Regina E. Mayer ◽  
Francisca Maurer ◽  
Stuart R. Stone ◽  
Brian A. Hemmings
Keyword(s):  
Transcriptional Regulation ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A

Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2

2016 ◽  
Vol 397 (6) ◽  
pp. 541-554 ◽  
Author(s):  
Panagiotis S. Athanasopoulos ◽  
Wright Jacob ◽  
Sebastian Neumann ◽  
Miriam Kutsch ◽  
Dirk Wolters ◽  
...  
Keyword(s):  
Hela Cells ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Sodium Selenate ◽  
Leucine Rich Repeat ◽  
Human Neuroblastoma ◽  
Interacting Protein ◽  
Cellular Degeneration ◽  
Phosphatase 2A

Abstract Mutations in the gene coding for the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) are the leading cause of genetically inherited Parkinson’s disease (PD). Two of the common found mutations are the R1441C and G2019S. In this study we identified protein phosphatase 2A (PP2A) as an interacting partner of LRRK2. We were able to demonstrate that the Ras of complex protein (ROC) domain is sufficient to interact with the three subunits of PP2A in human neuroblastoma SH-SY5Y cells and in HeLa cells. The alpha subunit of PP2A is interacting with LRRK2 in the perinuclear region of HeLa cells. Silencing the catalytic subunit of PP2A by shRNA aggravated cellular degeneration induced by the pathogenic R1441C-LRRK2 mutant expressed in neuroblastoma SH-SY5Y cells. A similar enhancement of apoptotic nuclei was observed by downregulation of the catalytic subunit of PP2A in cultured cortical cells derived from neurons overexpressing the pathogenic mutant G2019S-LRRK2. Conversely, pharmacological activation of PP2A by sodium selenate showed a partial neuroprotection from R1441C-LRRK2-induced cellular degeneration. All these data suggest that PP2A is a new interacting partner of LRRK2 and reveal the importance of PP2A as a potential therapeutic target in PD.

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