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To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5620; Fax: 404-727-2738; dpallas@emory.edu

Under agreements between Upstate Biotechnology Inc. (Millipore), Santa Cruz Biotechnologies, Stratagene, Inc., and Emory University, David Pallas is entitled to a share of sales royalty received by the University from these companies. In addition, this same author serves as a consultant to Millipore. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies.

We are grateful to Dr. William Hahn and The RNAi Consortium at the Broad Institute for lentiviral shRNA vectors and advice, to Dr. David M. Virshup for the HA-tagged Bα construct, to Dr. Eva Schmeltz for HCT116 cells, to the German Gene Trap Consortium for the LCMT-1+/- embryonic stem cells, to David Martin and colleagues in the Emory Transgenic facility for help with the LCMT-1 knock-out, to William Dalton for advice on caspase assays, and to Anita Corbett and Jennifer Jackson for critical reading of the manuscript.

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Research Funding:

This work was supported by NCI, National Institutes of Health Grant CA57327 (toD. C. P.) and Predoctoral Fellowship Award CA 1236402(toJ. A. L.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

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Journal Title:

Journal of Biological Chemistry

Volume:

Volume 282, Number 42

Publisher:

, Pages 30974-30984

Type of Work:

Article | Final Publisher PDF

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 Bα 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 Bα regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2ABαAC heterotrimers. Treatment with a pancaspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Bα knockdown. LCMT-1 knockdown cells and Bα knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Bα are important for spindle checkpoint. Treatment of LCMT-1 and Bα 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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© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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