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J. Biol. Chem., Vol. 283, Issue 51, 35834-35844, December 19, 2008

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Requirements for Protein Phosphorylation and the Kinase Activity of Polo-like Kinase 1 (Plk1) for the Kinetochore Function of Mitotic Arrest Deficiency Protein 1 (Mad1)^*

Ya-Hui Chi, Kerstin Haller, Michael D. Ward, O. John Semmes, Yan Li, and Kuan-Teh Jeang¹

From the Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Microbiology and Molecular Cell Biology, Center for Biomedical Proteomics, Eastern Virginia Medical School, Norfolk, Virginia 23501

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr⁶⁸⁰, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.

Received for publication, June 30, 2008 , and in revised form, October 14, 2008.

^* This work was authored, in whole or in part, by National Institutes of Health staff. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4.

¹ Supported through intramural funding from the NIAID, National Institutes of Health. To whom correspondence should be addressed: Bldg. 4, Rm. 306, 9000 Rockville Pike, Bethesda, MD 20892-0460. Tel.: 301-496-6680; Fax: 301-480-3686; E-mail: kj7e{at}nih.gov.

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