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J. Biol. Chem., Vol. 280, Issue 27, 25611-25620, July 8, 2005

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Nuclear Localization and Mitogen-activated Protein Kinase Phosphorylation of the Multifunctional Protein CAD^*

Frederic D. Sigoillot, Damian H. Kotsis, Valerie Serre, Severine M. Sigoillot, David R. Evans, and Hedeel I. Guy¶

From the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201 and Université Paris 7, 75743 Paris Cedex 15, France

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.

Received for publication, April 26, 2005 , and in revised form, May 12, 2005.

^* This work was supported by National Institutes of Health Grants GM/CA60371 and GM47399. 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.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1502; Fax: 313-577-2765; E-mail: hguy{at}cmb.biosci.wayne.edu.

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