Endotoxin (LPS) stimulates 4E-BP1/PHAS-I phosphorylation in macrophages.
BACKGROUND
Translational control of cytokine production in endotoxin (LPS)-stimulated macrophages is poorly characterized but likely important. An early step in protein translation is engagement of mRNA by eukaryotic initiation factor 4E (eIF-4E). Translation initiation can be prevented by small 4E-binding proteins (4E-BP1 or PHAS-I) which must be phosphorylated in order to disengage eIF-4E. We examined whether LPS alters 4E-BP1 phosphorylation in macrophages.


METHODS
Elicited rat peritoneal macrophages and Raw 264.7 macrophages were treated with signal transduction inhibitors and then LPS. Cells were harvested and equal protein amounts were electrophoresed (SDS-PAGE). Western blots (WB) were developed with 4E-BP1 antibody. Alternatively cell lysates were exposed to 7-methyl GTP Sepharose beads in order to isolate the cap-binding protein eIF-4E. The relative amounts of 4E-BP1 associated with eIF-4E were then determined by WB.


RESULTS
Macrophage 4E-BP1 is phosphorylated upon stimulation by LPS as evidenced by the appearance of a more slowly migrating gamma (hyperphosphorylated) band on gel electrophoresis. Inhibition of both the p42/p44 MAPK pathway (PD 98059) and the p38 MAPK pathway (SB 203580) failed to alter LPS-induced 4E-BP1 phosphorylation. Rapamycin (FRAP/mTOR inhibitor) blocked 4E-BP1 phosphorylation causing a predominance of the alpha (hypophosphorylated) band. This was confirmed further by 7-methyl-GTP Sepharose isolation of eIF-4E with which 4E-BP1 coprecipitates.


CONCLUSIONS
LPS stimulates 4E-BP1 phosphorylation in macrophages through FRAP/mTOR signaling. This pathway may contribute to the translational control of cytokine gene expression in macrophages.
Potter
M W
MW
Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Shah
S A
SA
Elbirt
K K
KK
Callery
M P
MP
D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E; D005419:Flavonoids / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D008954:Models, Biological; D010448:Peptide Initiation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D020123:Sirolimus / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D005419:Flavonoids; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011725:Pyridines; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus