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J. Biol. Chem., Vol. 280, Issue 14, 14085-14096, April 8, 2005

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The 14-3-3 Proteins of Trypanosoma brucei Function in Motility, Cytokinesis, and Cell Cycle^*

Masahiro Inoue, Yasuo Nakamura¶, Kouichi Yasuda¶, Natsumi Yasaka||, Tatsuru Hara, Achim Schnaufer**, Kenneth Stuart**, and Toshihide Fukuma

From the Department of Parasitology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan, ^||Excel Japan Group, Shinjuku, Tokyo, Japan, and ^**Seattle Biomedical Research Institute, Seattle, Washington 98109-5219

The cDNAs for two isoforms (I and II) of the 14-3-3 proteins have been cloned and functionally characterized in Trypanosoma brucei. The amino acid sequences of isoforms I and II have 47 and 50% identity to the human isoform, respectively, with important conserved features including a potential amphipathic groove for the binding of phosphoserine/phosphothreonine-containing motifs and a nuclear export signal-like domain. Both isoforms are abundantly expressed at approximately equal levels (1–2 x 10⁶ molecules/cell) and localized mainly in the cytoplasm. Knockdown by induction of double-stranded RNA of isoform I and/or II in both bloodstream and procyclic forms resulted first in a reduction of cell motility and then significant reduction in cell growth rates and morphological changes; the changes include aberrant numbers of organelles and abnormal shapes and sizes that mimic phenotypes produced by various cytokinesis inhibitors. Morphological and fluorescence-activated cell sorting analysis of the cell cycle suggested that isoforms I and II might play important roles in nuclear (G₂-M transition) and cell (M-G₁ transition) division. These findings indicate that the 14-3-3 proteins play important roles in cell motility, cytokinesis, and the cell cycle.

Received for publication, November 1, 2004 , and in revised form, December 13, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB059827 and AB066565.

^* 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 an additional table and three figures.

^¶ These two authors contributed equally to this work.

To whom correspondence may be addressed. Fax: 81-942-31-0344; E-mail: inouedna{at}med.kurume-u.ac.jp. To whom correspondence may be addressed. Fax: 81-942-31-0344.

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