Purification and identification of a novel subunit of protein serine/threonine phosphatase 4.

The catalytic subunit of protein serine/threonine phosphatase 4 (PP4C) has greater than 65% amino acid identity to the catalytic subunit of protein phosphatase 2A (PP2AC). Despite this high homology, PP4 does not appear to associate with known PP2A regulatory subunits. As a first step toward characterization of PP4 holoenzymes and identification of putative PP4 regulatory subunits, PP4 was purified from bovine testis soluble extracts. PP4 existed in two complexes of approximately 270-300 and 400-450 kDa as determined by gel filtration chromatography. The smaller PP4 complex was purified by sequential phenyl-Sepharose, Source 15Q, DEAE2, and Superdex 200 gel filtration chromatographies. The final product contained two major proteins: the PP4 catalytic subunit plus a protein that migrated as a doublet of 120-125 kDa on SDS-polyacrylamide gel electrophoresis. The associated protein, termed PP4R1, and PP4C also bound to microcystin-Sepharose. Mass spectrometry analysis of the purified complex revealed two major peaks, at 35 (PP4C) and 105 kDa (PP4R1). Amino acid sequence information of several peptides derived from the 105 kDa protein was utilized to isolate a human cDNA clone. Analysis of the predicted amino acid sequence revealed 13 nonidentical repeats similar to repeats found in the A subunit of PP2A (PP2AA). The PP4R1 cDNA clone engineered with an N-terminal Myc tag was expressed in COS M6 cells and PP4C co-immunoprecipitated with Myc-tagged PP4R1. These data indicate that one form of PP4 is similar to the core complex of PP2A in that it consists of a catalytic subunit and a "PP2AA-like" structural subunit.

Carboxymethylation of nuclear protein serine/threonine phosphatase X.

Specific rabbit polyclonal antibodies against peptides corresponding to the highly homologous protein serine/threonine phosphatase 2A and X catalytic subunits (PP2A/C and PPX/C respectively) were used to investigate the cellular and subcellular distribution of PP2A/C and PPX/C, as well as their methylation state. Immunoblots of rat tissue extracts revealed a widespread distribution of these enzymes but particularly high levels of PP2A/C and PPX/C in brain and testes respectively. In addition, immunoblots of subcellular fractions and immunocytochemical analyses of rat brain sections demonstrated that PPX/C is predominantly localized to the nucleus, whereas PP2A/C is largely cytoplasmic. Treatment of nuclear extracts with alkali resulted in increased PPX/C immunoreactivity to a polyclonal antibody directed against the C-terminus; no change in PPX immunoreactivity was observed using an antibody against an internal peptide. Alkali treatment of brain and liver cytosolic and nuclear extracts did not change the molecular mass or the isoelectric point of PPX/C. Furthermore, tritiated PPX/C was immunoprecipitated from COS cell extracts incubated with the methyl donor S-adenosyl-l-[methyl-3H]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 Hemmings (1994) J. Biol. Chem. 269, 16311-16317]. Together, our results indicate that the PPX catalytic subunit is a predominantly nuclear phosphatase and is methylated at its C-terminus.