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␣ 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.Protein phosphatase 2A (PP2A), the most abundant serine/ threonine-specific phosphatase in mammals, plays a role in many fundamental cellular processes, including cell division (7, 30), signal transduction (36), gene expression (52), and Drosophila development (34). The PP2A holoenzyme consists of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit, which together form the core enzyme to which one of several B subunits is bound (8, 38). The A and C subunits both exist as two isoforms (␣ and ␤) (19, 63), whereas the B subunits fall into three families called B, BЈ, and BЉ, which are unrelated to each other by protein sequence. The B family has three members, B␣, B␤, and B␥, each with a molecular mass of around 55 kDa (18,33,41); the BЈ family consists of numerous isoforms and splice variants, whose molecular masses range from 54 to 68 kDa (10, 35, 57, 69); and the BЉ family has two members, which have molecular masses of 72 and 130 kDa and are splice variants of the same gene (20). The combination of these subunits can give rise to a large number of PP2A variants, which ...

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine͞threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.

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.

The protein phosphatase 2A (PP2A) holoenzyme consists of a catalytic subunit, C, and two regulatory subunits, A and B. The PP2A core enzyme is composed of subunits A and C. Both the holoenzyme and the core enzyme are similarly abundant in heart tissue. Transgenic mice were generated expressing high levels of a dominant negative mutant of the A subunit (A delta 5) in the heart, skeletal muscle, and smooth muscle that competes with the endogenous A subunit for binding the C subunit but does not bind B subunits. We found that the ratio of core enzyme to holoenzyme was increased in A delta 5-expressing hearts. Importantly, already at day 1 after birth, A delta 5-transgenic mice had an increased heart weight-to-body weight ratio that persisted throughout life. Echocardiographic analysis of A delta 5-transgenic hearts revealed increased end-diastolic and end-systolic dimensions and decreased fractional shortening. In addition, the thickness of the septum and of the left ventricular posterior wall was significantly reduced. On the basis of these findings, we consider the heart phenotype of A delta 5-transgenic mice to be a form of dilated cardiomyopathy that frequently leads to premature death.