Circumventing Embryonic Lethality with Lcmt1 Deficiency: Generation of Hypomorphic Lcmt1 Mice with Reduced Protein Phosphatase 2A Methyltransferase Expression and Defects in Insulin Signaling

Abstract

Protein phosphatase 2A (PP2A), the major serine/threonine phosphatase in eukaryotic cells, is a heterotrimeric protein composed of structural, catalytic, and targeting subunits. PP2A assembly is governed by a variety of mechanisms, one of which is carboxyl-terminal methylation of the catalytic subunit by the leucine carboxyl methyltransferase LCMT1. PP2A is nearly stoichiometrically methylated in the cytosol, and although some PP2A targeting subunits bind independently of methylation, this modification is required for the binding of others. To examine the role of this methylation reaction in mammalian tissues, we generated a mouse harboring a gene-trap cassette within intron 1 of Lcmt1. Due to splicing around the insertion, Lcmt1 transcript and LCMT1 protein levels were reduced but not eliminated. LCMT1 activity and methylation of PP2A were reduced in a coordinate fashion, suggesting that LCMT1 is the only PP2A methyltransferase. These mice exhibited an insulin-resistance phenotype, indicating a role for this methyltransferase in signaling in insulin-sensitive tissues. Tissues from these animals will be vital for the in vivo identification of methylation-sensitive substrates of PP2A and how they respond to differing physiological conditions.

PP2A Inhibition Is a Common Event in Colorectal Cancer and Its Restoration Using FTY720 Shows Promising Therapeutic Potential

“…Interestingly, we observed a significant reduction of PP2A activity in 18 out of the 21 samples analyzed ( Table S2). Because the 1D6 antibody against PP2A has been previously reported to recognize the demethylated fraction of PP2A (21)(22), we performed these experiments with a second antibody against full-length PP2A (FL-309), and similar results were observed in all cases except for P4 who was Figs. S2 and S3).…”

“…PP2A assays were carried out with two different antibodies against PP2A (1D6 and FL-309) that showed similar results in all cases except in P4 (PP2A inhibition only with FL-309). Although the FL-309 antibody recognizes the full-length PP2A, the 1D6 antibody has been reported to preferentially recognize the demethylated fraction of PP2A (21)(22), and this could be an explanation for the discrepancy observed in this case P4 between the PP2A assays performed with these antibodies (Supplementary Table S2). To determine the molecular mechanisms responsible of this PP2A inhibition in colorectal cancer, we sequenced PPP2R1B but no missense mutations were found in our cohort.…”

H2O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells

“…Methylation of PP2A is catalyzed by a specific S-adenosyl methionine (SAM)-dependent methyltransferase, the leucine carboxyl methyltransferase 1 (LCMT1) (Stanevich et al, 2014), while the removal of the carboxyl methyl group is catalyzed by a specific PP2A methyl esterase, protein phosphatase methylesterase 1 (PME-1) (Xing et al, 2008). Current research has shown that PP2A methylation is involved in a wide spectrum of physiological and pathological conditions, including, but not limited to, insulin sensitivity (MacKay et al, 2013), immune function (Abolhassani et al, 2008;Kranias et al, 2010), neurodegenerative disease (Vafai and Stock, 2002;Lee et al, 2011;Sontag et al, 2013), and cancer (Guenin et al, 2008). Although the functional significance of PP2A methylation has been known, how PP2A methylation relate to PP2A downstream signaling pathways is not fully understood.…”

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

“…Previously, we reported that homozygous global knockout of LCMT-1 in mice via a gene trap approach causes embryonic lethality, indicating that LCMT-1 is essential for mouse development (18). Subsequently, it was reported that global hypomorphic knockdown of LCMT-1 resulted in some viable homozygous LCMT-1 knockdown mice that showed decreased glucose tolerance and increased glucose-stimulated insulin secretion, consistent with a possible insulin resistance phenotype (29). However, no other phenotypes were reported.…”