Leucine Carboxyl Methyltransferase 1 (LCMT-1) Methylates Protein Phosphatase 4 (PP4) and Protein Phosphatase 6 (PP6) and Differentially Regulates the Stable Formation of Different PP4 Holoenzymes

Abstract

The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.

Global loss of leucine carboxyl methyltransferase-1 causes severe defects in fetal liver hematopoiesis

“…Based on this finding, LCMT-1 was proposed to be a master regulator of these phosphatases, which share ϳ60% sequence identity, including the C-terminal leucine, the site of methylation by LCMT-1. Similar to the case for PP2A, LCMT-1 differentially regulates the formation and function of different PP4 heterotrimers, but effects on PP6 heterotrimers were not seen (19).…”

“…Recently, LCMT-1 was shown to be the methyltransferase responsible for methylation of not just PP2A but of all three members of the PP2A subfamily of serine/threonine protein phosphatases, PP2A, PP4, and PP6 (19). Based on this finding, LCMT-1 was proposed to be a master regulator of these phosphatases, which share ϳ60% sequence identity, including the C-terminal leucine, the site of methylation by LCMT-1.…”

Protein Serine/Threonine Phosphatases: Keys to Unlocking Regulators and Substrates

“…Studies showed enhanced activity of LCMT-1 with the PP2A AC dimer versus LCMT-1 with the isolated PP2A catalytic subunit, positing a role for the scaffolding A subunit in promoting PP2A methyl-esterification (216). LCMT-1 is the major enzyme that catalyzes methyl-esterification of not only PP2A but also PP4 and PP6 catalytic subunits (217). On the basis of results with PP2A, we might expect LCMT-1 to promote the assembly of multisubunit complexes for multiple PPP types.…”

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

“…Knockdown of LCMT1 would lead to the loss of activity of PP2A and cell death [22]. Moreover, LCMT1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function [23]. As an expected PP2A activator, LCMT1 overexpression induced the dephosphorylation of a majority (74.72%) of peptides in HEK293 cells.…”