DIFFERENTIAL METHYLATION AND ALTERED CONFORMATION OF CYTOPLASMIC AND NUCLEAR FORMS OF PROTEIN PHOSPHATASE 2A DURING CELL-CYCLE PROGRESSION

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activ ation (and deactivation) of the various holoenzymes to initiate approp riate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies agains t both PP2Ac and PR65 show that these proteins are ubiquitously locali zed in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP 2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in t he nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity alb eit the total amounts of neither PP2Ac nor PR65 in each compartment al ters significantly during the cell cycle. Our results imply that diffe rential methylation of PP2Ac occurs at the G(0)/G(1) and G(1)/S bounda ries. Specifically a demethylated form of PP2Ac is found in the cytopl asm of G(1) cells, and in the nucleus of S and G(2) cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G(0)/G(1) and G(1)/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no spe cific association with the condensed chromatin.