CARBOXYMETHYLATION OF NUCLEAR-PROTEIN SERINE THREONINE PHOSPHATASE-X/

Specific rabbit polyclonal antibodies against peptides corresponding t o the highly homologous protein serine/threonine phosphatase 2A and X catalytic subunits (PP2A/C and PPX/C respectively) were used to invest igate the cellular and subcellular distribution of PP2A/C and PPX/C, a s well as their methylation state. Immunoblots of rat tissue extracts revealed a widespread distribution of these enzymes but particularly h igh levels of PP2A/C and PPX/C in brain and testes respectively. In ad dition, immunoblots of subcellular fractions and immunocytochemical an alyses of rat brain sections demonstrated that PPX/C is predominantly localized to the nucleus, whereas PP2A/C is largely cytoplasmic. Treat ment of nuclear extracts with alkali resulted in increased PPX/C immun oreactivity to a polyclonal antibody directed against the C-terminus; no change in PPX immunoreactivity was observed using an antibody again st an internal peptide. Alkali treatment of brain and liver cytosolic and nuclear extracts did not change the molecular mass or the isoelect ric point of PPX/C. Furthermore, tritiated PPX/C was immunoprecipitate d from COS cell extracts incubated with the methyl donor S-adenosyI-L- [methyl-H-3]methionine. Thus the increase in immunoreactivity probably results from removal of a carboxymethyl group from PPX/C, as has been shown previously for PP2A/C [Favre, Zolnierowicz, Turowski and Hemmin gs (1994) J. Biol. Chem. 269, 16311-16317]. Together, our results indi cate that the PPX catalytic subunit is a predominantly nuclear phospha tase and is methylated at its C-terminus.