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J. Biol. Chem., Vol. 279, Issue 46, 47688-47698, November 12, 2004

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Construction of a Cyclin D1-Cdk2 Fusion Protein to Model the Biological Functions of Cyclin D1-Cdk2 Complexes^*

Anna Chytil, Mary Waltner-Law¶||, Robert West, David Friedman**, Mary Aakre, Dana Barker, and Brian Law

From the Vanderbilt-Ingram Cancer Center, the Department of Cancer Biology, the ^¶Department of Molecular Physiology and Biophysics, and the ^**Vanderbilt Proteomics Laboratory, Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232

Cyclin D1 is frequently overexpressed in human breast cancers, and cyclin D1 overexpression correlates with poor prognosis. Cyclin D1-Cdk2 complexes were previously observed in human breast cancer cell lines, but their role in cell cycle regulation and transformation was not investigated. This report demonstrates that Cdk2 in cyclin D1-Cdk2 complexes from mammary epithelial cells is phosphorylated on the activating phosphorylation site, Thr¹⁶⁰. Furthermore, cyclin D1-Cdk2 complexes catalyze Rb phosphorylation on multiple sites in vitro. As a model to investigate the biological and biochemical functions of cyclin D1-Cdk2 complexes, and the mechanisms by which cyclin D1 activates Cdk2, a cyclin D1-Cdk2 fusion gene was constructed. The cyclin D1-Cdk2 fusion protein expressed in epithelial cells was phosphorylated on Thr¹⁶⁰ and catalyzed the phosphorylation of Rb on multiple sites in vitro and in vivo. Kinase activity was not observed if either the cyclin D1 or Cdk2 domain was mutationally inactivated. Mutational inactivation of the cyclin D1 domain prevented activating phosphorylation of the Cdk2 domain on Thr¹⁶⁰. These results indicate that the cyclin D1 domain of the fusion protein activated the Cdk2 domain through an intramolecular mechanism. Cells stably expressing the cyclin D1-Cdk2 fusion protein exhibited several hallmarks of transformation including hyperphosphorylation of Rb, resistance to TGF-induced growth arrest, and anchorage-independent proliferation in soft agar. We propose that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 and demonstrate that the cyclin D1-Cdk2 fusion protein is a useful model to investigate the biological functions of cyclin D1-Cdk2 complexes.

Received for publication, May 27, 2004 , and in revised form, August 5, 2004.

^* This work was supported by National Institutes of Health Grant R01-CA93651 (to B. L.), Vanderbilt-Ingram Comprehensive Cancer Center Support Grant CA68485, and the Frances Williams Preston Laboratories of the T. J. Martell Foundation. 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.

^|| Present address: Dept. of Pharmacology and Therapeutics, Shands Cancer Center, University of Florida, Academic RSCH BLDG, Rm. R5-126, 1600 SW Archer Rd., P.O. Box 100267, Gainesville, FL 32610.

To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, Shands Cancer Center, University of Florida, Academic RSCH BLDG, Rm R5-126, 1600 SW Archer Rd., P.O. Box 100267, Gainesville, FL 32610. E-mail: bklaw{at}pharmacology.ufl.edu.

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