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J. Biol. Chem., Vol. 282, Issue 42, 30974-30984, October 19, 2007

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

From the Department of Biochemistry, Winship Cancer Center, and Biochemistry, Cell, and Developmental Biology Program, Emory University School of Medicine, Atlanta, Georgia 30322

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 B 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 B regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A_BAC heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or B knockdown. LCMT-1 knockdown cells and B knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and B are important for spindle checkpoint. Treatment of LCMT-1 and B 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.

Received for publication, June 13, 2007 , and in revised form, August 8, 2007.

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

¹ 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. 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; E-mail: dpallas{at}emory.edu.

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