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.

L-isoaspartyl (D-aspartyl) O-methyltransferase deficient mice (Pcmt1−/−) accumulate isomerized aspartyl residues in intracellular proteins until their death due to seizures at approximately 45 days. Previous studies have shown that these mice have constitutively activated insulin signaling in their brains, and that these brains are 20–30% larger than those from age-matched wild-type animals. To determine whether insulin pathway activation and brain enlargement is responsible for the fatal seizures, we administered wortmannin, an inhibitor of the phosphoinositide 3-kinase that catalyzes an early step in the insulin pathway. Oral wortmannin reduced the average brain size in the Pcmt1−/− animals to within 6% of the wild-type DMSO administered controls, and nearly doubled the lifespan of Pcmt1−/− at 60% survival of the original population. Immunoblotting revealed significant decreases in phosphorylation of Akt, PDK1, and mTOR in Pcmt1−/− mice and Akt and PDK1 in wild-type animals upon treatment with wortmannin. These data suggest activation of the insulin pathway and its resulting brain enlargement contributes to the early death of Pcmt1−/− mice, but is not solely responsible for the early death observed in these animals.

Protein Phosphatase 2 A (PP2A) is a highly conserved, broad specificity, heterotrimeric serine/threonine phosphatase involved in nearly all cytosolic processes. PP2A control is manifested through subunit composition, small molecule and peptide inhibitors as well as post‐translational modifications. One such modification, methylation of the C‐terminal leucine by LCMT1, regulates PP2A subunit assembly, specifically Bα subunit binding, and thus substrate specificity. In this study we generate a mouse homozygous for an Lcmt1 gene‐trap cassette, yet expressing reduced Lcmt1 transcript and protein. Full length Lcmt1 transcript is achieved through alternative splicing around the genetrap cassette in a tissue specific manner. Reduced LCMT1 correlates with a concomitant reduction of PP2A methylation and an increase in PP2A demethylation confirming LCMT1 as the main PP2A methyltransferase. Lcmt1 homozygous gene‐trapped mice were born in a non‐mendelian ratio suggesting embryonic lethality. Adult Lcmt1 hypomorphic animals display decreased glucose tolerance and increased glucose‐stimulated insulin secretion suggesting an insulin resistance phenotype. Our results indicate that PP2A methylation has a role in propagation of the insulin‐signaling cascade, possibly through altered subunit composition of PP2A.This work was supported by the National Institutes of Health (GM026020)