Purification of Porcine Brain Protein Phosphatase 2A Leucine Carboxyl Methyltransferase and Cloning of the Human Homologue^,

Abstract

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

“…Thus, gPP2A-C localizes to specific PDR, which accompany only the intracellular portions of the corresponding axonemes, rather than to the flagellar axonemes themselves. Similar results were observed with permeabilized intact cells, regardless of the method of fixation and also with the methylation sensitive antibody against PP2A-C (Ab 299/309 ) [29] (data not shown). We did not detect gPP2A-C in the nuclei or in the cytosol.…”

“…For detection with the methylation sensitive antibody, filters were treated for 10 min with 0.2 M KOH prior to blocking of the membrane; control membranes were untreated. Filters were incubated with either the methylation sensitive (Ab 299/309 ) [29] or the non-methylation sensitive anti-PP2A-C polyclonal antibody [25], the rabbit polyclonal antibody against PDI-2 [30] as a protein loading control or the mouse monoclonal antibody against CWP-1. As a control for specificity, the KVTRRTPDYFL peptide used for immunization [25] or the Giardia C-terminal PP2A-C peptide HISRRVPDYFL (GenScript Corporation) was pre-incubated with the non-methylation sensitive PP2A-C antibody for 30 min.…”

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Carboxymethylation [41] occurs on the free carboxyl group of the C-terminal Leu309 residue and is catalyzed by the S-adenosylmethionine-dependent LCMT1 (leucine carboxyl methyltransferase 1) [42,43]. It is estimated that 70%-90% of PP2A C is methylated in vivo [44].…”

“…Methylation selectively affects PP2A T55 holoenzyme assembly In contrast to phosphorylation, PP2A C methylation does not significantly affect PP2A activity directly [42,50]. However, a variety of experimental approaches both in yeast and mammalian cells demonstrate that PP2A C methylation has a crucial role in B-type-subunit binding, specifically in the recruitment of PR55/B subunits [20][21][22][23]44,[51][52][53][54][55].…”

Structure of Protein Phosphatase Methyltransferase 1 (PPM1), a Leucine Carboxyl Methyltransferase Involved in the Regulation of Protein Phosphatase 2A Activity

“…Methylation of the mammalian PP2A has been shown to be carried out by a specific methyltransferase (leucine carboxyl methyltransferase 1, also known as LCMT1) (3). Two homologues have been found in Saccharomyces cerevisiae, PPM1 and PPM2, which share, respectively, 30 and 26% sequence identity to the mammalian PP2A methyltransferase.…”