title: Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit.
abstract: Methylation of the C-terminal leucine residue (Leu309) of protein serine/threonine phosphatase 2A catalytic subunit (PP2AC) is known to regulate catalytic activity in vitro, but the functional consequence(s) of this post-translational modification in the context of the cell remain unclear. Alkali-induced demethylation of PP2AC in purified PP2A heterotrimer (ABalphaC), but not in purified PP2A heterodimer (AC), indicated that a larger fraction of PP2AC is carboxymethylated in ABalphaC than in AC. To explore the role of Leu309 in PP2A holoenzyme assembly, epitope-tagged PP2A catalytic subunit (HA-PP2A) and a mutant of HA-PP2A containing an alanine residue in place of Leu309 (HA-PP2A-L309A) were transiently expressed in COS cells. Both recombinant proteins exhibited serine/threonine phosphatase activity when immunoisolated from COS cell extracts. HA-PP2A, but not HA-PP2A-L309A, was carboxymethylated in vitro. A chromatographic analysis of cell extracts indicated that most endogenous PP2AC and HA-PP2A were co-eluted with the A and Balpha regulatory subunits of PP2A, whereas most HA-PP2A-L309A seemed to elute with the A subunit as a smaller complex or, alternatively, as free catalytic (C) subunit. The A subunit co-immunoisolated with both tagged proteins; however, substantially less Balpha subunit co-immunoisolated with HA-PP2A-L309A than with HA-PP2A. These results demonstrate that the reversibly methylated C-terminal leucine residue of PP2AC is important for Balpha regulatory subunit binding. Furthermore, the results provide evidence for an interrelationship between PP2AC carboxymethylation and PP2A holoenzyme assembly.
authors: Department of Pharmacology, Room 424 MRB1, Vanderbilt University Medical Center, Nashville, TN 37232-6600, USA.
authors: J C
authors: JC
authors: Bryant
authors: R S
authors: RS
authors: Westphal
authors: B E
authors: BE
authors: Wadzinski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D020134:Catalytic Domain; D017931:DNA Primers; D007930:Leucine / Q000378:metabolism; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054648:Protein Phosphatase 2
chemical_list: D017931:DNA Primers; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D007930:Leucine