The highly acidic C-terminal region of the yeast initiation factor subunit 2 α (eIF-2 α) contains casein kinase phosphorylation sites and is essential for maintaining normal regulation of GCN4

Abstract Regulation of the effective activity of eukaryotic initiation factor 2 (eIF-2) in protein synthesis is known to involved phosphorylation of its α subunit. Two mammalian enzymes, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI), phosphorylate Ser-51 of the α subunit, thereby inhibiting the exchange of bound nucleotides on, and thus the recycling of, eIF-2. In Saccharomyces cerevisiae, the equivalent serine seems to be phosphorylated by the GCN2 protein kinase, which is activated by amino acid starvation. However, in the present paper we show that this is not the only site of phosphorylation in yeast eIF-2 α. We report the preparation of recombinant yeast eIF-2 α from Escherichia coli and its use in in vitro phosphorylation studies. Mammalian HCR and dsI are shown to phosphorylate specifically Ser-51 of yeast eIF-2 α, whereas extracts from yeast cells do not. Instead, at least one of three serine residues in the acidic C-terminal region of this protein is phosphorylated by fractions of yeast possessing casein kinase activities 1 and 2. A triple Ser → Ala mutant form of yeast eIF-2 α was found to be no longer phosphorylated by either of the yeast (or mammalian) casein kinase activities in vitro. Isoelectric focusing of yeast extracts confirmed that the mutated sites normally act as sites of phosphorylation in vivo. The same mutant was used to show that the three sites have no essential function under normal physiological conditions in yeast. In contrast, deletion of the 13 amino acid long C-terminal region of eIF-2 α, including the three phosphorylation sites, led to derepression of GCN4 in vivo. Thus removal of the short, highly acidic C-terminal region of eIF-2 α has the same regulatory effect on translational (re)initiation as phosphorylation of the Ser-51 residue of the wild-type protein. This result provides new insight into the role of eIF-2 α activity in the regulation of translational (re-) initiation.

[1]  A. Cigan,et al.  Mutations at a Zn(II) finger motif in the yeast elF-2β gene alter ribosomal start-site selection during the scanning process , 1988, Cell.

[2]  V. M. Pain.  Initiation of protein synthesis in mammalian cells. , 1986, The Biochemical journal.

[3]  C. Proud,et al.  Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the alpha subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor. , 1987, European journal of biochemistry.

[4]  E. Krebs,et al.  Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. , 1991, The Journal of biological chemistry.

[5]  S. Ochoa.  Regulation of protein synthesis initiation in eucaryotes. , 1983, Archives of biochemistry and biophysics.

[6]  J. McCarthy,et al.  A fully modular vector system for the optimization of gene expression in Escherichia coli. , 1991, Plasmid.

[7]  J. Hershey,et al.  Translational control in mammalian cells. , 1991, Annual review of biochemistry.

[8]  Gary M. Hathaway,et al.  [33] Casein kinase I , 1983 .

[9]  A. Hinnebusch.  A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae , 1985, Molecular and cellular biology.

[10]  A. Kamath,et al.  Role of yeast elongation factor 3 in the elongation cycle. , 1989, Journal of Biological Chemistry.

[11]  L. Pinna,et al.  Endogenous phosphate acceptor proteins for rat liver cytosolic casein kinases. , 1981, The Journal of biological chemistry.

[12]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[13]  B. Chait,et al.  Isolation and properties of YCK2, a Saccharomyces cerevisiae homolog of casein kinase-1. , 1993, Archives of biochemistry and biophysics.

[14]  E. W. Jones.  Proteinase mutants of Saccharomyces cerevisiae. , 1977, Genetics.

[15]  L. Pinna,et al.  Structure and properties of casein kinase-2 from Saccharomyces cerevisiae. A comparison with the liver enzyme. , 1986, European journal of biochemistry.

[16]  Mick F. Tuite,et al.  Post-Transcriptional Control of Gene Expression , 1990, NATO ASI Series.

[17]  J. McCarthy,et al.  Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem‐loops in the mRNA leader , 1993, Molecular microbiology.

[18]  T. Donahue,et al.  Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae , 1994, Molecular and cellular biology.

[19]  B. Sefton,et al.  Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. , 1989, Analytical biochemistry.

[20]  M. Mathews,et al.  Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI , 1989, Molecular and cellular biology.

[21]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

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

[23]  J. Sayers,et al.  5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. , 1988, Nucleic acids research.

[24]  F. Eckstein,et al.  Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. , 1986, Nucleic acids research.

[25]  G. Welsh,et al.  Evidence that insulin activates casein kinase 2 in rat epididymal fat-cells and that this may result in the increased phosphorylation of an acid-soluble 22 kDa protein. , 1991, The Biochemical journal.

[26]  H. Blöcker,et al.  Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. , 1987, Gene.

[27]  G. Natsoulis,et al.  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. , 1987, Methods in enzymology.

[28]  C. Proud,et al.  Phosphorylation of protein synthesis initiation factor-2. Identification of the site in the alpha-subunit phosphorylated in reticulocyte lysates. , 1990, Biochimica et biophysica acta.