Phosphorylation of eukaryotic protein synthesis initiation factor 4E by insulin-stimulated protamine kinase.
Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent Km for eIF-4E was about 1.2 and 50 microM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and < or = 5% activity with eIF-4ES209A and eIF-4ET210A, respectively, and eIF-4ES209A was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser209 and Thr210, that the hydroxyl group or phosphorylation of Thr210 is necessary for cPK to act on Ser209, and that Ser209 phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.
Makkinje
A
A
Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, Hershey 17033, USA.
Xiong
H
H
Li
M
M
Damuni
Z
Z
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary; D004789:Enzyme Activation; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism*; D054648:Protein Phosphatase 2
D018076:DNA, Complementary; D039561:Eukaryotic Initiation Factor-4E; D007328:Insulin; D010448:Peptide Initiation Factors; D010748:Phosphopeptides; D006656:Protamine Kinase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2