Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth.
The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.
Basu
Uttiya
U
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.
Si
Kausik
K
Deng
Haiteng
H
Maitra
Umadas
U
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D047388:Casein Kinases; D002455:Cell Division / Q000502:physiology; D017124:Conserved Sequence; D039642:Eukaryotic Initiation Factors; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008322:Mammals; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry; D012269:Ribosomal Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015003:Yeasts / Q000254:growth & development* / Q000378:metabolism
D002352:Carrier Proteins; C109284:EIF6 protein, human; D039642:Eukaryotic Initiation Factors; D007381:Intermediate Filament Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C491609:TIF6 protein, S cerevisiae; D012694:Serine; D011494:Protein Kinases; D047388:Casein Kinases