scholarly journals Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells.

1997 ◽  
Vol 17 (3) ◽  
pp. 1692-1701 ◽  
Author(s):  
E Kremmer ◽  
K Ohst ◽  
J Kiefer ◽  
N Brewis ◽  
G Walter
Keyword(s):  
Monoclonal Antibodies ◽  
Gene Families ◽  
Regulatory Subunits ◽  
Core Enzyme ◽  
C Terminus ◽  
C Subunit ◽  
Phosphatase 2A ◽  
A Subunit ◽  
B Subunits ◽  
In Cells

Protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit, C, and two regulatory subunits, A and B. The A subunit is rod shaped and consists of 15 nonidentical repeats. According to our previous model, the B subunit binds to repeats 1 through 10 and the C subunit binds to repeats 11 through 15 of the A subunit. Another form of PP2A, core enzyme, is composed only of subunits A and C. It is generally believed that core enzyme does not exist in cells but is an artifact of enzyme purification. To study the structure and relative abundance of different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitopes in repeat 1 near the N terminus and immunoprecipitated free A subunit and core enzyme but not holoenzyme. Another antibody, 6G3, recognized an epitope in repeat 15 at the C terminus and precipitated only the free A subunit. Monoclonal antibodies against a peptide corresponding to the N-terminal 11 amino acids of the A alpha subunit (designated 6F9) precipitated free A subunit, core enzyme, and holoenzyme. 6F9, but not 5H4, recognized holoenzymes containing either B, B', or B" subunits. These results demonstrate that B subunits from three unrelated gene families all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoenzyme. By sequential immunoprecipitations with 5H4 or 1A12 followed by 6F9, core enzyme and holoenzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and they exhibited the expected specificities towards phosphorylase a and retinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holoenzyme, core enzyme represented at least one-third of the total PP2A. We conclude that core enzyme is an abundant form in cells rather than an artifact of isolation. The biological implications of this finding are discussed.

scholarly journals Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit

1999 ◽  
Vol 339 (2) ◽  
pp. 241-246 ◽  
Author(s):  
Jeffrey C. BRYANT ◽  
Ryan S. WESTPHAL ◽  
Brian E. WADZINSKI
Keyword(s):  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Post Translational Modification ◽  
Regulatory Subunits ◽  
Leucine Residue ◽  
Cell Extracts ◽  
C Subunit ◽  
Phosphatase 2A ◽  
A Subunit

Methylation of the C-terminal leucine residue (Leu309) of protein serine/threonine phosphatase 2A catalytic subunit (PP2AC) is known to regulate catalytic activity in vitro, but the functional consequence(s) of this post-translational modification in the context of the cell remain unclear. Alkali-induced demethylation of PP2AC in purified PP2A heterotrimer (ABαC), but not in purified PP2A heterodimer (AC), indicated that a larger fraction of PP2AC is carboxymethylated in ABαC than in AC. To explore the role of Leu309 in PP2A holoenzyme assembly, epitope-tagged PP2A catalytic subunit (HA-PP2A) and a mutant of HA-PP2A containing an alanine residue in place of Leu309 (HA-PP2A-L309A) were transiently expressed in COS cells. Both recombinant proteins exhibited serine/threonine phosphatase activity when immunoisolated from COS cell extracts. HA-PP2A, but not HA-PP2A-L309A, was carboxymethylated in vitro. A chromatographic analysis of cell extracts indicated that most endogenous PP2AC and HA-PP2A were co-eluted with the A and Bα regulatory subunits of PP2A, whereas most HA-PP2A-L309A seemed to elute with the A subunit as a smaller complex or, alternatively, as free catalytic (C) subunit. The A subunit co-immunoisolated with both tagged proteins; however, substantially less Bα subunit co-immunoisolated with HA-PP2A-L309A than with HA-PP2A. These results demonstrate that the reversibly methylated C-terminal leucine residue of PP2AC is important for Bα regulatory subunit binding. Furthermore, the results provide evidence for an interrelationship between PP2AC carboxymethylation and PP2A holoenzyme assembly.

scholarly journals Binding Specificity of Protein Phosphatase 2A Core Enzyme for Regulatory B Subunits and T Antigens

1999 ◽  
Vol 73 (1) ◽  
pp. 839-842 ◽  
Author(s):  
Ralf Ruediger ◽  
Katherine Fields ◽  
Gernot Walter
Keyword(s):  
Amino Acids ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Binding Specificity ◽  
T Antigen ◽  
T Antigens ◽  
Core Enzyme ◽  
Phosphatase 2A ◽  
A Subunit ◽  
B Subunits

ABSTRACT The core enzyme of protein phosphatase 2A is composed of a regulatory subunit A and a catalytic subunit C. It is controlled by three types of regulatory B subunits (B, B′, and B") and by tumor (T) antigens, which are unrelated by sequence but bind to overlapping regions on the A subunit. To find out whether the different B subunits and T antigens bind to identical or distinct amino acids of the A subunit, mutants were generated and their abilities to bind B subunits and T antigens were tested. We found that some amino acids are involved in the binding of all types of B subunits, whereas others are specifically involved in the binding of one or two types of B subunits. T-antigen-binding specificity does not correlate with that of a particular type of B subunit.

Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition

2020 ◽  
Vol 13 (616) ◽  
pp. eaax6490 ◽  
Author(s):  
Ingrid E. Frohner ◽  
Ingrid Mudrak ◽  
Stephanie Kronlachner ◽  
Stefan Schüchner ◽  
Egon Ogris
Keyword(s):  
Monoclonal Antibody ◽  
Protein Phosphatase ◽  
Phosphorylation Sites ◽  
Regulatory Subunits ◽  
C Terminus ◽  
C Subunit ◽  
Phosphatase 2A ◽  
Phosphatase Assay ◽  
Commercial Antibodies ◽  
Modified Forms

The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies—including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit—are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits. The tested antibodies preferentially recognized the nonmethylated form of the enzyme, and they did not coimmunoprecipitate trimeric holoenzymes containing the regulatory subunits B or B′, an issue that precludes their use to monitor PP2A holoenzyme activity. Furthermore, some of the antibodies also recognized the phosphatase PP4, demonstrating a lack of specificity for PP2A. Together, these findings suggest that reinterpretation of the data generated by using these reagents is required.

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 Selection of Protein Phosphatase 2A Regulatory Subunits Is Mediated by the C Terminus of the Catalytic Subunit

2007 ◽  
Vol 282 (37) ◽  
pp. 26971-26980 ◽  
Author(s):  
Sari Longin ◽  
Karen Zwaenepoel ◽  
Justin V. Louis ◽  
Stephen Dilworth ◽  
Jozef Goris ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Regulatory Subunits ◽  
C Terminus ◽  
Phosphatase 2A ◽  
Selection Of

scholarly journals Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

2020 ◽  
Vol 295 (17) ◽  
pp. 5654-5668 ◽  
Author(s):  
Chian Ju Jong ◽  
Ronald A. Merrill ◽  
Emily M. Wilkerson ◽  
Laura E. Herring ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Second Messengers ◽  
Phosphorylation Site ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Endogenous Expression ◽  
Phosphatase 2A ◽  
B Subunits

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B′/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge–swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge–swap pair and the endogenous catalytic subunit. Using B′δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal–regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B′δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.

scholarly journals Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen

2001 ◽  
Vol 12 (1) ◽  
pp. 185-199 ◽  
Author(s):  
Xing Xian Yu ◽  
Xianxing Du ◽  
Carlos S. Moreno ◽  
Richard E. Green ◽  
Egon Ogris ◽  
...  
Keyword(s):  
Tumor Antigen ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Methylation Status ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
C Subunit ◽  
Phosphatase 2A ◽  
Carboxy Terminal

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Bα subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Bα subunit binding without greatly affecting methylation, indicating that Bα subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Bα subunit but not the amount that could be coimmunoprecipitated with Aα subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Bα subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Bα subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

scholarly journals Effects of carboxyl-terminal methylation on holoenzyme function of the PP2A subfamily

2020 ◽  
Vol 48 (5) ◽  
pp. 2015-2027
Author(s):  
Isha Nasa ◽  
Arminja N. Kettenbach
Keyword(s):  
Substrate Specificity ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Phosphoprotein Phosphatases ◽  
Phosphatase 2A ◽  
Adenosyl Methionine ◽  
Carboxyl Terminal ◽  
Different Levels ◽  
In Cells

Phosphoprotein Phosphatases (PPPs) are enzymes highly conserved from yeast and human and catalyze the majority of the serine and threonine dephosphorylation in cells. To achieve substrate specificity and selectivity, PPPs form multimeric holoenzymes consisting of catalytic, structural/scaffolding, and regulatory subunits. For the Protein Phosphatase 2A (PP2A)-subfamily of PPPs, holoenzyme assembly is at least in part regulated by an unusual carboxyl-terminal methyl-esterification, commonly referred to as ‘methylation’. Carboxyl-terminal methylation is catalyzed by Leucine carboxyl methyltransferase-1 (LCMT1) that utilizes S-adenosyl-methionine (SAM) as the methyl donor and removed by protein phosphatase methylesterase 1 (PME1). For PP2A, methylation dictates regulatory subunit selection and thereby downstream phosphorylation signaling. Intriguingly, there are four families of PP2A regulatory subunits, each exhibiting different levels of methylation sensitivity. Thus, changes in PP2A methylation stoichiometry alters the complement of PP2A holoenzymes in cells and creates distinct modes of kinase opposition. Importantly, selective inactivation of PP2A signaling through the deregulation of methylation is observed in several diseases, most prominently Alzheimer's disease (AD). In this review, we focus on how carboxyl-terminal methylation of the PP2A subfamily (PP2A, PP4, and PP6) regulates holoenzyme function and thereby phosphorylation signaling, with an emphasis on AD.

scholarly journals Effects of Carbon Source on Expression of Fo Genes and on the Stoichiometry of the c Subunit in the F1Fo ATPase of Escherichia coli

1998 ◽  
Vol 180 (12) ◽  
pp. 3205-3208 ◽  
Author(s):  
Randy A. Schemidt ◽  
Jun Qu ◽  
James R. Williams ◽  
William S. A. Brusilow
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Proton Channel ◽  
Gene Encoding ◽  
Nonfermentable Carbon Source ◽  
C Subunit ◽  
Metabolic Conditions ◽  
A Subunit ◽  
Membrane Bound ◽  
In Cells

ABSTRACT Expression of the genes for the membrane-bound Fosector of the Escherichia coli F1Foproton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric Fo proton channel. Transcriptional and translational lacZ fusions to the promoter and to two Fo genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of uncB, encoding the a subunit of Fo, are higher than during growth on glucose. In contrast, translation of the uncE gene, encoding the c subunit of Fo, is higher during growth on glucose than during growth on succinate. Translation rates of both uncB anduncE change as culture density increases, but transcription rates do not. Quantitation of the c stoichiometry shows that more c subunits are assembled into the F1Fo ATPase in cells grown on glucose than in cells grown on succinate. E. coli therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.

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