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


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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    • "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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    • "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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