Ilse Stevens

The protein phosphatase 2A phosphatase activator is a novel peptidyl-prolyl cis/trans-isomerase.

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 (PPIase) family. The PPIase activity of PTPA correlates with its activating activity since both are stimulated by the presence of Mg2+ATP, and a PTPA mutant (Δ208–213) with 400-fold less activity in the activation reaction of PP2A also showed almost no PPIase activity. The point mutant Asp205 → Gly (in Ypa1) identified this amino acid as essential for both activities. Moreover, PTPA dissociates the inactive form from the complex with the PP2A methylesterase. Finally, Pro190 in the catalytic subunit of PP2A (PP2AC) could be identified as the target Pro isomerized by PTPA/Mg2+ATP since among the 14 Pro residues present in 12 synthesized peptides representing the microenvironments of these prolines in PP2AC, only Pro190 could be isomerized by PTPA/Mg2+ATP. This Pro190 is present in a predicted loop structure near the catalytic center of PP2AC and, if mutated into a Phe, the phosphatase is inactive and can no longer be activated by PTPA/Mg2+ATP.

An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator.

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.

Specific interactions of PP2A and PP2A-like phosphatases with the yeast PTPA homologues, Ypa1 and Ypa2

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.