Leucine Carboxyl Methyltransferase-1 Is Necessary for Normal Progression through Mitosis in Mammalian Cells

Emory University, Atlanta, Georgia, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2007; 282(42):30974-84. DOI: 10.1074/jbc.M704861200
Source: PubMed

ABSTRACT Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the Balpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A Balpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(BalphaAC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Balpha knockdown. LCMT-1 knockdown cells and Balpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Balpha are important for spindle checkpoint. Treatment of LCMT-1 and Balpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.

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    • "PP2A is a family of abundant brain Ser/Thr phosphatases that collectively participate in nearly all aspects of neuronal homeostasis. Of particular interest, biogenesis of major PP2A/Bα isoforms that are primary brain enzymes that dephosphorylate Tau, is critically influenced by LCMT1-mediated PP2Ac methylation (Lee and Pallas, 2007; Sontag et al., 2008). Decreased PP2A methylation and PP2A/Bα expression levels correlate with enhanced tau phosphorylation in several mouse models of altered one-carbon metabolism Reviewed in Sontag and Sontag (2014) and in diabetic mice (Papon et al., 2013). "
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    ABSTRACT: Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr (-/-) and aged heterozygous Mthfr (+/-) mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr (+/+) mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr (+/-) mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer's disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD.
    Frontiers in Aging Neuroscience 08/2014; 6:214. DOI:10.3389/fnagi.2014.00214 · 4.00 Impact Factor
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    • "The level of PP2A methylation was previously shown to vary during cell cycle37. LCMT-1 knockdown attenuated cell cycle progression and caused cell death38,39, so did loss of PP2A holoenzymes40,41,42. Whether PP2Ac can be activated by alternative mechanisms that lead to nonspecific pTyr phosphatase activity and cause cell death in the absence of PTPA remains to be determined. "
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    ABSTRACT: Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.Cell Research advance online publication 8 October 2013; doi:10.1038/cr.2013.138.
    Cell Research 10/2013; 24(2). DOI:10.1038/cr.2013.138 · 12.41 Impact Factor
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    • "In an attempt to elucidate the role of LCMT1 and PP2A methylation in higher organisms, we report the generation of an Lcmt1 hypomorphic mouse model, detailing the biochemical as well as the phenotypic effects of reduced LCMT1 activity. Although disrupting Lcmt1 was previously found to be lethal in an Lcmt1 gene-trap mouse [40], in this report we demonstrate partial LCMT1 activity in mice with a distinct Lcmt1 gene trap mutation. In these mice, splicing around the insertional mutation leads to the production of intact Lcmt1 transcripts and allows homozygous mice to survive embryonic development. "
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    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.
    PLoS ONE 06/2013; 8(6):e65967. DOI:10.1371/journal.pone.0065967 · 3.23 Impact Factor
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