We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

The protein phosphatase 2A (PP2A) phosphatase activator (PTPA) is an essential protein involved in the regulation of PP2A and the PP2A-like enzymes. In this study we demonstrate that PTPA and its yeast homologues Ypa1 and Ypa2 can induce a conformational change in some model substrates. Using these model substrates in different assays with and without helper proteases, this isomerase activity is similar to the isomerase activity of FKBP12, the human cyclophilin A, and one of its yeast homologs Cpr7 but dissimilar to the isomerase activity of Pin1. However, neither FKBP12 nor Cpr7 can reactivate the inactive form of PP2A. Therefore, PTPA belongs to a novel peptidyl-prolyl cis/trans-isomerase (

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B/PR61, and B؆/PR72). We identified two functional and highly conserved Ca 2؉ -binding EF-hand motifs in human B؆/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca 2؉ to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca 2؉ with different affinities. Ca 2؉ induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca 2؉ on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2A T72 ) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca 2؉ on PP2A T72 activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

To elucidate the specific biological role of the yeast homologues of PTPA (phosphatase 2A phosphatase activator), Ypa1 and Ypa2 (where Ypa stands for yeast phosphatase activator), in the regulation of PP2A (protein phosphatase 2A), we investigated the physical interaction of both Ypa proteins with the catalytic subunit of the different yeast PP2A-like phosphatases. Ypa1 interacts specifically with Pph3, Sit4 and Ppg1, whereas Ypa2 binds to Pph21 and Pph22. The Ypa1 and Ypa2 proteins do not compete with Tap42 (PP2A associating protein) for binding to PP2A family members. The interaction of the Ypa proteins with the catalytic subunit of PP2A-like phosphatases is direct and independent of other regulatory subunits, implicating a specific function for the different PP2A-Ypa complexes. Strikingly, the interaction of Ypa2 with yeast PP2A is promoted by the presence of Ypa1, suggesting a positive role of Ypa1 in the regulation of PP2A association with other interacting proteins. As in the mammalian system, all yeast PP2A-like enzymes associate as an inactive complex with Yme (yeast methyl esterase). Ypa1 as well as Ypa2 can reactivate all these inactive complexes, except Pph22-Yme. Ypa1 is the most potent activator of PP2A activity, suggesting that there is no direct correlation between activation potential and binding capacity.