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Abstract
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.
Highlights
From the Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Milton S
Insulin-stimulated protamine kinase and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping
Phosphopeptide Mapping-As a first step to examine the sites modified on eIF-4E by cytosolic Protamine Kinase (cPK), and to relate the results to insulin action, we examined the tryptic phosphopeptides derived from 32P-Iabeled elF-4E prepared by incubation with cPK or PKC
Summary
14824-14828, 1995 Printed in U.S.A. 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. 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. All the other insulinstimulated protein kinases examined displayed little or no activity with eIF-4E (23-25), raising the possibility that cPK may contribute to the insulin-stimulated phosphorylation of eIF-4E (23). The results are consistent with the idea that cPK contributes to the insulin-stimulated phosphorylation of elF-4E and concomitant activation of protein synthesis, but indicate an important role for PP2A in this insulin response
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