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. 2013 Jun 20;8(6):e65967.
doi: 10.1371/journal.pone.0065967. Print 2013.

Circumventing embryonic lethality with Lcmt1 deficiency: generation of hypomorphic Lcmt1 mice with reduced protein phosphatase 2A methyltransferase expression and defects in insulin signaling

Kennen B MacKay  1 , Yiping Tu, Stephen G Young, Steven G Clarke
Affiliations

Circumventing embryonic lethality with Lcmt1 deficiency: generation of hypomorphic Lcmt1 mice with reduced protein phosphatase 2A methyltransferase expression and defects in insulin signaling

Kennen B MacKay et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genomic confirmation of Lcmt1−/− animals.
Panel A: Southern blot showing a single band of 9.8 kDa corresponding to the predicted AflII restriction product. No other bands were seen at other sizes in Lcmt1−/− gel lane or in the gel lanes of WT DNA samples. Panel B: Schematic of Lcmt1+/+ and Lcmt1−/− genomic DNA showing the position of the gene-trap insertion, PCR primers, and AflII restriction sites flanking the gene trap cassette. Panel C: Primers flanking the gene-trap cassette insertion site (WT primers) amplify an 1899-bp product when the gene trap is absent; primers inside the gene trap cassette (KO primers) amplify a 524-bp product when the gene trap is present (see “Methods”). When the 8.6-kb gene-trap cassette is present, WT primer amplification is prevented.
Figure 2
Figure 2. Initial intercrosses of Lcmt1+/− mice yield lower-than-expected numbers of homozygous offspring.
Pups were counted on the day they were born and genotyped at 18 days of age.
Figure 3
Figure 3. Lcmt1 transcript quantitation in wild-type and Lcmt1−/− mouse tissues.
Panel A: Lcmt1 transcript levels from five tissues, measured using primers flanking exons 1 and 2, and exons 2 and 3 as described in the “Methods” section, were normalized to β-2 microglobulin transcript levels measured in the same tissue. ΔCt values are displayed above each column. Values were averaged from tissues prepared from three wild-type and three knockout mice; all qPCR determinations were performed in duplicate. Errors bars show the standard deviation. Panel B: Lcmt1−/− transcript levels as a percent of Lcmt1+/+ transcript levels. Error bars represent the standard deviation. Asterisks indicate where the decrease in Lcmt1−/− transcript levels compared to those of wild-type mice are significant at a p value of less than 0.05 by the Student's t-test. Panel C: the upper schematic shows the generation of full-length Lcmt1 mRNA in wild-type mice while the middle schematic shows the generation of a truncated transcript in Lcmt1−/− mice. The lower schematic shows how alternative splicing can skip over the gene trap cassette to produce a full-length mRNA transcript.
Figure 4
Figure 4. Quantitation of LCMT1 levels in tissues of Lcmt1−/− animals compared to wild-type controls.
Panel A: Representative Western blots of tissue extracts with antibodies against LCMT1. Antibodies to β-actin were used as a loading control. All samples were electrophoresed on the same gel along with protein ladder standards. Panel B: Summary of results from experiments with extracts from three wild-type and three Lcmt1−/− mice. Densitometry was used to normalize LCMT1 levels to actin. The ratio of Lcmt1−/− to Lcmt1+/+ signals were then plotted with the error bars indicating the standard deviation of three experiments. In each case, extracts of wild-type and knockout tissues were electrophoresed on the same gel as shown in Panel A. Asterisks indicate significant decreases in expression (p value less than 0.05) by Student's t-test.
Figure 5
Figure 5. Quantitation of the steady state methylation level of PP2A in tissues of Lcmt1−/− and Lcmt1+/+ mice.
Panel A–D: Tissue extracts from four wild-type and four knockout animals were treated either with 0.1 M NaOH for 1 min at room temperature to cleave methyl esters followed by addition of 0.1 M HCl and 0.5 M Tris-HCl, pH 7.4 to neutralize the solution (NaOH +), or a previously neutralized buffer containing the aforementioned solutions as a control (NaOH −). Polypeptides from these extracts were then separated by SDS-PAGE using 17-well, 4–12% Bis-Tris NuPAGE gels (Invitrogen), transferred to PVDF membranes, and incubated with clone 1D6 antibodies selective for the demethylated catalytic subunit of PP2A, and antibodies to GAPDH as a loading control. These procedures are described in detail in “Methods”. Panels E–H: The percent of the PP2A catalytic subunit that is demethylated was calculated as described by Yu et al. . The columns represent the average ± the standard deviation for the four Lcmt1−/− and four Lcmt1+/+ mice shown in Panel A–D. Statistical significance at the level of p less than 0.05 was determined by the Student's t-test and is denoted by the asterisk.
Figure 6
Figure 6. Quantification of in vitro PP2A methylation.
Extracts from Lcmt1−/− and Lcmt1+/+ mice were incubated with [3H]AdoMet as described in the “Methods.” The radioactive peak of [3H]methyl esters corresponding to the position of the PP2A catalytic subunit at ∼36 kDa was quantified after the background (from a parallel lane of molecular weight standards) was subtracted. Each column represents the mean of three independent experiments utilizing tissue extracts from three Lcmt1−/− and three Lcmt1+/+ mice. Error bars represent the standard deviation. Asterisks indicate statistically-significant differences between extracts from wild-type and knockdown mice (p value less than 0.05) with the Student's t-test.
Figure 7
Figure 7. Decreased glucose tolerance in Lcmt1−/− mice.
Male and female mice were fasted overnight and administered 2 g of glucose orally per kg body mass followed by measurement of whole blood glucose levels as described in the “ Methods .” Panel A: Results from three wild-type and three knockout male mice. Error bars represent standard deviation; asterisks indicate statistically-significant differences between wild-type and knockout mice (p value less than 0.05) with the Student's t-test. Panel B: Results as in panel A but using 3 wild-type and 3 knockout female mice.
Figure 8
Figure 8. Male Lcmt1−/− animals display increased glucose-stimulated insulin secretion.
Three wild-type and three knockout mice were fasted overnight and administered 2 g of glucose per kg of body mass, followed by measurement of plasma insulin levels as described in the “ Methods .” Error bars represent standard deviations; asterisks indicate statistically-significant differences between wild-type and knockout mice (p value less than 0.05) with the Student's t-test.
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