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Molecular & Cellular Proteomics 4:993-1001, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis^*

Zizheng Dong^,, Randy J. Arnold^¶, Youyun Yang, Myung Hee Park^|, Petra Hrncirova^¶, Yehia Mechref^¶, Milos V. Novotny^¶ and Jian-Ting Zhang^,**

From the Department of Pharmacology and Toxicology, Indiana University Cancer Center and Walther Oncology Center/Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202, ^¶ Department of Chemistry, Indiana University, Bloomington, Indiana 47405, and ^| NIDCR, National Institutes of Health, Bethesda, Maryland 20892

L-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G₁ phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G₁ phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [³⁵S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

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