Increasing the Ratio of PP2A Core Enzyme to Holoenzyme Inhibits Tat-Stimulated HIV-1 Transcription and Virus Production

Abstract

We demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoenzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) core enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A core and holoenzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, A delta 5, which binds the C but not the B subunit. This resulted in an increase in the amount of core enzyme and a decrease in the amount of holoenzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant A delta 5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant A delta 5. These results demonstrate that the balance between PP2A holoenzyme and core enzyme is important for HIV-1 gene expression and virus production.

Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the Aβ subunit gene

“…We analysed eight mutants of the Ab subunit, six of which were found in lung carcinoma and two in colon carcinoma. The binding studies were carried out using an in vitro binding assay that faithfully re¯ects in vivo binding as demonstrated for several mutants in di erent systems, including the yeast two-hybrid system (McCright and Virshup, 1995), mammalian cells in tissue culture (Ruediger et al, 1997), and mice (Brewis et al, 2000). Two N-terminal mutants (P65S and DE344 ± E388) showed strongly decreased B''/PR72 binding, while two C-terminal mutants (V448A and V545A) showed strongly decreased B''/PR72 and C binding consistent with the previous ®nding that B subunits cooperate with the C subunit in binding to the A subunit.…”

The Formation and Activity of PP2A Holoenzymes Do Not Depend on the Isoform of the Catalytic Subunit

“…For transfection, 1.5 ϫ 10 6 293 cells were plated per 10-cm dish, grown for 24 h, and transfected with a total of 3.9 g of plasmid DNA using 30 l of LipofectAMINE and 20 l of PLUS reagent following Invitrogen's instructions. Transfection conditions were optimized 1) for high and similar expression levels of EE-tagged A␣ and A␤ as determined by Western blotting with anti-EE antibodies, 2) for high expression of co-transfected tagged B or C subunits, and 3) for high transfection efficiency as determined by staining with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal) of fixed cells co-transfected with a ␤-galactosidase vector (18). 48 h after transfection, the cells were harvested either with SDS-PAGE sample buffer (2% SDS, 100 mM 1,4-dithiothreitol (DTT), 60 mM Tris-HCl, pH 6.8, 10% glycerol, and 0.01% bromphenol blue) for Western blotting or with TX-100 buffer (0.5% TX-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 3 mM MgCl 2 , 1 mM DTT, and 50 M leupeptin for immunoprecipitation.…”

“…In panel a, the exposure times are 20 min for A␣ EE (lanes 1-21), 16 h for A␣ EE (lanes [22][23][24][25][26][27][28], and 5 min for the Bs and Cs. In panel b, the exposure times are 30 s for A␤ EE (lanes 1-10), 20 min for A␤ EE (lanes [11][12][13][14][15][16][17][18][19][20], 5 min for the Bs and Cs (lanes 1-5), and 30 min for the Bs and Cs (lanes 6 -20). IP, immunoprecipitation.…”

Reduced expression of the A? subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the A? and A? subunit genes

“…Equal amounts of protein were separated on 10% SDS polyacrylamide gels and transferred to PVDF membrane (Immobilon-P, Millipore). To detect the A, B and C subunits, the following primary antibodies were used: rat monoclonal anti-A subunit (6G3), 30 rabbit anti-B␣ subunit produced against the peptide KGAVDDDVAEADY, 31 mouse monoclonal anti-C subunit from Marc Mumby and rabbit anti-A␤ subunit produced against the peptide MAGASELGTGPGK coupled to keyhole limpet hemacyanin with glutaraldehyde. Appropriate secondary antibodies coupled to horseradish peroxidase (Jackson ImmunoResearch) were used as well as Renaissance chemiluminescence reagent (NEN Life Science Products).…”