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,Toshihide Fukuma

doi:10.1074/jbc.m412336200

Abstract

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 tau 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 10(6) 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.

Highlights

14-3-3 proteins are a group of dimeric acidic proteins with a relative molecular mass of 30 kDa that bind to phosphoserine/ phosphothreonine-containing sequences. 14-3-3 proteins are well conserved and exist in all lower and higher eukaryotes
The disease is spread by the bite of the tsetse fly, in which procyclic forms (PCF)1 proliferate and differentiate into bloodstream forms (BSF), the life cycle stage that proliferates in the mammalian host
When the expression of either 14-3-3 gene was knocked down in BSF cells, we observed isoform-specific reduction in the respective 14-3-3 mRNA level but reduction in protein levels of both isoforms. This unusual phenomenon in BSF cells could be due to post-translational regulation; we assume that dimerization of 14-3-3 isoforms is required for their stabilization in BSF but not PCF cells, heterodimers are formed in PCF cells

Summary

Introduction

14-3-3 proteins are a group of dimeric acidic proteins with a relative molecular mass of 30 kDa that bind to phosphoserine/ phosphothreonine-containing sequences. 14-3-3 proteins are well conserved and exist in all lower and higher eukaryotes. 14-3-3 proteins are crucial for cell survival signaling through binding to proteins such as Raf-1, proapoptotic proteins BAD and BAX, transcription factor FKHR, and cell cycle phosphatase Cdc 14-3-3 proteins in Saccharomyces cerevisiae are associated with upstream activation of the Ras/ mitogen-activated protein kinase signaling pathway (STE 20), a basic helix-loop-helix transcription factor (RTG3), nutrientregulated transcription factors (MSN2 and MSN4), and cell cycle-dependent filaments in the nucleus (FIN 1) (for a review, see Ref. 9). 14-3-3 proteins in Schizosaccharomyces pombe bind to cell cycle-dependent phosphatase (Cdc25) and phospholipase C [9]. Yeast 14-3-3 proteins are functionally interchangeable with the plant and mammalian isoforms [9], suggesting genetic conservation of 14-3-3 molecules in these species. To elucidate the functions of 14-3-3 in PCF and BSF cells, we generated and characterized 14-3-3 isoform-specific Tet-inducible double-stranded RNA (dsRNA)-expressing cell clones

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Results

Conclusion