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. 2016 Sep 30;291(40):21008-21019.
doi: 10.1074/jbc.M116.739920. Epub 2016 Aug 9.

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

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Free PMC article

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

Juyeon Hwang et al. J Biol Chem. .
Free PMC article

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.

Keywords: Lcmt-1; PP4; PP6; methyltransferase; protein complex; protein methylation; protein phosphatase 2 (PP2A); protein phosphorylation; signaling.

Figures

FIGURE 1.
FIGURE 1.
PP4c and PP6c are highly methylated by LCMT-1 in MEFs. A and B, testing methylation-sensitive PP4c and PP6c antibodies. PP4c and PP6c peptides corresponding to their 16 C-terminal amino acid residues including a methylated C-terminal leucine were synthesized, and equal amounts of methylated and demethylated PP4c (A) and PP6c (B) peptides on nitrocellulose membranes were probed by Western blotting with methylation-sensitive PP4c (A) and PP6c (B) antibodies. C, preadsorption of methylation-sensitive PP4c antibody with PP2Ac peptide. PP4c methylation-sensitive antibody (anti-PP4c) was pre-adsorbed with PP2Ac C-terminal peptide for 1 h rocking at 4 °C or not preadsorbed and then used for immunoblotting of strip blots along with PP2Ac antibody (control). Lane 1, immunoblotting of lysate with PP2Ac antibody. Lane 2, immunoblotting of identical lysate with non-preadsorbed PP4c antibody. Lane 3, immunoblotting of the same lysate with PP4c antibody preadsorbed with PP2Ac peptide. Although the unabsorbed PP4c antibody detected both endogenous PP2Ac and PP4c (lane 2), preadsorption of PP4c antibody with PP2Ac C-terminal peptide removed PP2Ac signal without substantially affecting PP4c detection. +, 20 μg of PP2Ac peptide. Results are representative of three different experiments. D and E, examples of methylation assays for PP4c and PP6c on MEFs performed as described under “Experimental Procedures.” Equal amounts of cell lysates from LCMT-1 WT and KO MEFs were treated with (+) or without (−) base, and then proteins were resolved by SDS-PAGE. Western blotting was performed using methylation-sensitive PP4c (D) or methylation-sensitive PP6c (E) antibodies. α-Tubulin (α-Tub) was immunoblotted for a loading control. The low ratio of signal in the minus (−) lane compared with the plus (+) lane in WT cells in both panel D and panel E indicates that both PP4c and PP6c are highly methylated. The nearly equivalent levels of signal in the minus and plus lanes in KO cells indicates that LCMT-1 is the major methyltransferase for both PP4c and PP6c. In addition, comparison of PP6c levels in the plus lanes shows that the level of PP6c is increased in KO cells relative to WT cells. Of note, the C subunits of all PP2A subfamily phosphatases migrate sometimes as singlets and sometimes as doublets (e.g. for PP6 in panel E) (Refs. and unpublished data); whether double or single bands are seen can vary for the same sample from gel to gel. F, quantification of the percent methylation of PP4c and PP6c in WT and KO cells. Error bars represent the S.D. of at least three independent experiments. **, p ≤ 0.01 when compared with WT using Student's t test.
FIGURE 2.
FIGURE 2.
PP2A B subunit/C subunit association is dramatically decreased in LCMT-1 KO MEFs. A and B, immunoprecipitation of PP2A B subunit from WT (+/+), hemizygous (+/−), and KO (−/−) LCMT-1 MEFs was performed using anti-B subunit monoclonal antibody, 2G9, cross-linked to Sepharose beads. Lysates (A) and immunoprecipitates (IP, B) were resolved by SDS-PAGE and analyzed by immunoblotting for B subunit (Bsub), PP2Ac (Csub), and actin. Actin serves as a loading control. C, beads only control. C, quantification of the relative association of PP2Ac with B subunit. Error bars represent S.D. of three independent experiments. **, p ≤ 0.01 when compared with WT using Student's t test.
FIGURE 3.
FIGURE 3.
BN-PAGE facilitates analysis of PP2A complexes in WT and LCMT-1 KO MEFs. A–C, two sets of WT and LCMT-1 KO MEFs originating from paired embryos from different litters (M1 and M2) were lysed in non-denaturing lysis buffer, and equal amounts (20 μg/well) of lysates were resolved by BN-PAGE. PP2A complexes were analyzed by immunoblotting (WB, Western blot) with antibodies against the B subunit (2G9) (A), total PP2Ac (B), or PP2A A subunit (C). Panel A: asterisk, methylation-dependent PP2A heterotrimer; filled arrows, smaller B subunit-containing bands; open arrow, high molecular weight complexes (doublet) containing PP2A B subunit. Panel B: asterisk, methylation-dependent PP2A heterotrimer; solid arrowhead, methylation-independent PP2A complex; open arrowhead, high molecular weight complex enhanced in KO and containing PP2Ac plus an unknown binding partner(s). Panel C: asterisk, methylation-dependent PP2A heterotrimer; solid arrowhead, methylation-independent PP2A complex. Data in panels A and C are representative of three experiments, whereas data in panel B are representative of four experiments. The apparent difference in the relative ratio of A subunit in the two bands in the WT lanes of panel C compared with PP2Ac in panel B may be due to the fact that panel C was obtained by reprobing of blots. The relative ratio of these two bands in Panel B probably reflects the true ratio of these PP2A complexes in MEF lysates.
FIGURE 4.
FIGURE 4.
Loss of PP2Ac methylation by LCMT-1 knock-out increases co-migration of α4 with PP2Ac. A, cell lysates from two sets (M1 and M2) of WT and LCMT-1 KO MEFs were prepared under non-denaturing conditions and resolved by BN-PAGE. The proteins were transferred to PVDF and probed for α4 by immunoblotting (WB, Western blot). Open arrowhead, α4 co-migrating with the PP2Ac bands noted by the same symbol in Fig. 3B and Fig. 4B; arrow, α4 complex that does not co-migrate with PP2A components; bracket, additional α4 bands. B, QBI293 cells expressing HA-tagged WT PP2Ac, HA-tagged ΔLeu-309 mutant PP2Ac, or empty vector (VC, vector control) were lysed in non-denaturing conditions, and cell lysates were resolved by BN-PAGE. Antibody against the HA tag (16B12) was used to identify complexes containing HA-tagged WT or ΔLeu-309 mutant PP2Ac. Asterisk, methylation-dependent PP2A heterotrimer; solid arrowhead, methylation-independent PP2A complex; arrow, free PP2Ac; open arrowhead, HA-tagged PP2Ac that co-migrates with α4 and increases with the loss of Leu-309. Data in panel A are representative of five experiments, whereas data in panel B are representative of two experiments.
FIGURE 5.
FIGURE 5.
Loss of LCMT-1 differentially affects different PP4 protein phosphatase complexes. A and B, the importance of LCMT-1 for stable PP4 and PP6 complex formation was investigated by BN-PAGE analysis of non-denatured lysates from two independent sets (M1 and M2) of WT and LCMT-1 KO MEFs followed by immunoblotting with antibodies against PP4c (A) and PP6c (B). A: asterisk, methylation-dependent PP4 complex; filled arrowhead, PP4 complex less affected by LCMT-1 loss; bracket, PP4c-containing bands increased in KO. B, filled arrowhead, the major PP6 complex. Data in each panel are representative of at least three experiments. WB, Western blot.
FIGURE 6.
FIGURE 6.
Immunoblotting analysis to investigate the PP4 regulatory subunit composition of the 300- and 450-kDa PP4 complexes. Nondenatured WT MEF cell lysates were analyzed by BN-PAGE and immunoblotting using antibodies against PP4c and PP4 regulatory subunits. WB Ab, immunoblotting antibody for that lane. Filled arrowhead, 450-kDa PP4 complex; asterisk, 300-kDa PP4 complex. PP4R2, PP4R3α, and PP4R3β comigrated with the 450-kDa PP4 band, whereas PP4R1 and a small amount of PP4R3α comigrated with the 300-kDa PP4 band. Data show representative results from at least two experiments.
FIGURE 7.
FIGURE 7.
BN-PAGE EMSA facilitates definitive identification of PP4 regulatory subunits in the 300- and 450-kDa PP4 complexes. A and B, nondenatured WT MEF cell lysates were incubated with the indicated PP4 regulatory subunit antibodies (PP4R Ab) before analysis by BN-PAGE and immunoblotting with anti-PP4c antibody. A, mobility shift of PP4 complexes by PP4R1 or PP4R2 antibodies. B, mobility shift of PP4 complexes by PP4R3α, PP4R3β, or both PP4R3α/β antibodies. Asterisk, 300-kDa PP4 band; filled arrowhead, 450-kDa PP4 band; brackets, mobility shifted PP4 complexes. Ab only, control samples with the indicated antibodies in lysis buffer. C and D, schematic of PP4 subunits identified in the 300-kDa (C) and 450-kDa (D) complexes. The data do not rule out the presence of additional proteins in these complexes. Experiments were repeated at least three times, and representative data are shown.
FIGURE 8.
FIGURE 8.
LCMT-1 loss causes differential effects on steady-state PP4 subunit levels and associations. A–C, PP4 regulatory subunit immunoprecipitates were prepared using cell lysates from WT and LCMT-1 KO MEFs, and then immunoprecipitates (IP) and lysates were resolved by SDS-PAGE. The relative association of PP4c with PP4R1 (A), PP4R2 (B), and PP4R3β (C) was analyzed by immunoblotting. PP4R3α antibody did not immunoprecipitate reliably so PP4c/PP4R3α association was not analyzed in these experiments. Immunoblotting for GAPDH in lysates was included as a loading control for lysates. D, quantification of the relative association of PP4c with PP4R1, PP4R2, and PP4R3β normalized to WT. E and F, quantification of the steady-state levels of PP4 regulatory subunits (E) and PP4c (F) in WT and LCMT-1 KO MEFs. Error bars represent S.D. of at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01.
FIGURE 9.
FIGURE 9.
Loss of LCMT-1 results in increased phosphorylation of HDAC3 on an activating site. A, WT and LCMT-1 KO MEF lysates were resolved by SDS-PAGE and analyzed by immunoblotting to determine the level of phospho-HDAC3 and total HDAC3. B, quantification of the relative amount of phospho-HDAC3 (Ser-424) normalized to total HDAC3 in WT and LCMT-1 KO MEFs. Error bars represent S.D. of four independent experiments with at least three independently derived WT and KO MEF populations. **, p ≤ 0.01.
FIGURE 10.
FIGURE 10.
LCMT-1 coordinately regulates the methylation and likely the function of the PP2A subfamily of protein phosphatases. Black arrows show previously established connections. Blue arrows indicate relationships established in this study. The pink arrow with a question mark indicates the open question of whether there are PP6 methylation-dependent complexes regulated by LCMT-1. If they do not exist, methylation of PP6c still might regulate PP6 function via another mechanism. In this model loss of LCMT-1 could lead to dysfunction of all three PP2A subfamily phosphatases, whereas in normal cells regulation of LCMT-1 might coordinately regulate these phosphatases in common (e.g. DNA repair) functions. Also based on this model, independent regulation of these phosphatases by LCMT-1 would likely require distinct colocalization or scaffolding of LCMT-1 with a particular PP2A subfamily phosphatase. SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine.

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