eIF2α Kinases Regulate Development through the BzpR Transcription Factor in Dictyostelium discoideum

Background A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system. Principal Findings Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. β-galactosidase production was used to examine translation from mRNA containing the bzpR 5′ UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no β-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5′ UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated. Conclusions The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5′ UTR.

[1]  R. Wek,et al.  Phosphorylation of eIF2 Facilitates Ribosomal Bypass of an Inhibitory Upstream ORF to Enhance CHOP Translation*♦ , 2011, The Journal of Biological Chemistry.

[2]  R. Bowman,et al.  eIF2α Kinases Control Chalone Production in Dictyostelium discoideum , 2011, Eukaryotic Cell.

[3]  A. Ivens,et al.  A new Dictyostelium prestalk cell sub-type , 2010, Developmental biology.

[4]  P. V. van Haastert,et al.  A new set of small, extrachromosomal expression vectors for Dictyostelium discoideum. , 2009, Plasmid.

[5]  Donghui Zhou,et al.  Phosphorylation of eIF2 Directs ATF5 Translational Control in Response to Diverse Stress Conditions* , 2008, Journal of Biological Chemistry.

[6]  M. Rai,et al.  Disruption of the ifkA and ifkB genes results in altered cell adhesion, morphological defects and a propensity to form pre-stalk O cells during development of Dictyostelium. , 2006, Differentiation; research in biological diversity.

[7]  D. Ron,et al.  Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response , 2004, The Journal of cell biology.

[8]  R. Wek,et al.  Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Y. Xiong,et al.  IfkA, a presumptive eIF2α kinase of Dictyostelium, is required for proper timing of aggregation and regulation of mound size , 2003, BMC Developmental Biology.

[10]  Peichuan Zhang,et al.  The PERK Eukaryotic Initiation Factor 2α Kinase Is Required for the Development of the Skeletal System, Postnatal Growth, and the Function and Viability of the Pancreas , 2002, Molecular and Cellular Biology.

[11]  P. Sarnow,et al.  Regulation of Internal Ribosomal Entry Site-mediated Translation by Phosphorylation of the Translation Initiation Factor eIF2α* , 2002, The Journal of Biological Chemistry.

[12]  T. E. Dever,et al.  Gene-Specific Regulation by General Translation Factors , 2002, Cell.

[13]  J. W. Brewer,et al.  PERK mediates cell-cycle exit during the mammalian unfolded protein response. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Schapira,et al.  Regulated translation initiation controls stress-induced gene expression in mammalian cells. , 2000, Molecular cell.

[15]  J. Sternfeld.  The anterior-like cells in Dictyostelium are required for the elevation of the spores during culmination , 1998, Development Genes and Evolution.

[16]  P. Martin,et al.  Thiamine deficiency decreases steady-state transketolase and pyruvate dehydrogenase but not alpha-ketoglutarate dehydrogenase mRNA levels in three human cell types. , 1998, The Journal of nutrition.

[17]  P. Martin,et al.  Conserved residues are functionally distinct within transketolases of different species. , 1996, Biochemistry.

[18]  J. Williams,et al.  The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination. , 1996, Development.

[19]  J. Williams,et al.  Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination. , 1993, Development.

[20]  A. Hinnebusch,et al.  Phosphorylation of initiation factor 2α by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast , 1992, Cell.

[21]  D. Cavener,et al.  Eukaryotic start and stop translation sites. , 1991, Nucleic acids research.

[22]  B. M. Jackson,et al.  Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control , 1991, Molecular and cellular biology.

[23]  T. Dingermann,et al.  Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum. , 1989, Gene.

[24]  V. Pathak,et al.  The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs , 1989, Molecular and cellular biology.

[25]  G. Thireos,et al.  Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase , 1988, Molecular and cellular biology.

[26]  R. Delude,et al.  Characterization of genes which are deactivated upon the onset of development in Dictyostelium discoideum. , 1987, Developmental biology.

[27]  C N David,et al.  Fate and regulation of anterior-like cells in Dictyostelium slugs. , 1982, Developmental biology.

[28]  M. Sussman,et al.  RNA IN CYTOPLASMIC AND NUCLEAR FRACTIONS OF CELLULAR SLIME MOLD AMEBAS , 1970, The Journal of cell biology.

[29]  F. Půta,et al.  Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivation of genes in Dictyostelium. , 1998, Folia biologica.

[30]  A. Hinnebusch,et al.  7 Translational Control of GCN4: Gene-specific Regulation by Phosphorylation of elF2 , 1996 .

[31]  M. Sussman.  Chapter 14 Biochemical and Genetic Methods in the Study of Cellular Slime Mold Development , 1966 .