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.
Kloeker
S
S
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Bryant
J C
JC
Strack
S
S
Colbran
R J
RJ
Wadzinski
B E
BE
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D002467:Cell Nucleus / Q000201:enzymology*; D006801:Humans; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008745:Methylation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D054648:Protein Phosphatase 2; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C080406:protein phosphatase 4