Phosphorylation of mammalian translation initiation factor 5 (eIF5) in vitro and in vivo

Nucleic Acids Res. 2002 Mar 1;30(5):1154-62. doi: 10.1093/nar/30.5.1154.

Abstract

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S*eIF3*AUG*Met-tRNA(f)*eIF2*GTP) and, acting as a GTPase activating protein, promotes the hydrolysis of bound GTP. We isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate purified bacterially expressed recombinant rat eIF5. Physical, biochemical and antigenic properties of this kinase identify it as casein kinase II (CK II). Mass spectrometric analysis of maximally in vitro phosphorylated eIF5 localized the major phosphorylation sites at Ser-387 and Ser-388 near the C-terminus of eIF5. These serine residues are embedded within a cluster of acidic amino acid residues and account for nearly 90% of the total in vitro eIF5 phosphorylation. A minor phosphorylation site at Ser-174 was also observed. Alanine substitution mutagenesis at Ser-387 and Ser-388 of eIF5 abolishes phosphorylation by the purified kinase as well as by crude reticulocyte lysates. The same mutations also abolish phosphorylation of eIF5 when transfected into mammalian cells suggesting that CK II phosphorylates eIF5 at these two serine residues in vivo as well.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Casein Kinase II
  • Eukaryotic Initiation Factor-5
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Initiation Factors / chemistry
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / metabolism*
  • Phosphorylation
  • Phosphoserine / metabolism
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Protein-Serine-Threonine Kinases / physiology*
  • Rabbits
  • Rats
  • Recombinant Proteins / metabolism
  • Reticulocytes / enzymology

Substances

  • Eukaryotic Initiation Factor-5
  • Peptide Initiation Factors
  • Recombinant Proteins
  • Phosphoserine
  • Casein Kinase II
  • Protein-Serine-Threonine Kinases