Translational regulation during activation of porcine peripheral blood lymphocytes: association and phosphorylation of the alpha and gamma subunits of the initiation factor complex eIF-4F.
Mature peripheral blood lymphocytes exist in a resting state both in vivo and when maintained in culture, exhibiting low translation rates consistent with their non-proliferative state. Previously we have shown that activation of these quiescent cells with either phorbol ester or concanavalin A leads to a rapid increase in the rate of protein synthesis and phosphate-labelling of initiation factor eIF-4 alpha [Morley, Rau, Kay and Pain (1993) Eur. J. Biochem. 218, 39-48]. We now show that neither the early enhanced translation rate nor the early increased phosphate-labelling of eIF-4 alpha requires the activity of the 70 kDa form of ribosomal protein S6 kinase. In addition, we demonstrate that eIF-4 gamma is phosphorylated in response to cell activation, an event which is correlated with phosphorylation of eIF-4 alpha and enhanced eIF-4F complex formation. In these studies, isoelectric focusing and immunoblot analysis of eIF-4 alpha indicate that phosphate-labelling of eIF-4 alpha following cell activation reflects a modest increase in steady-state phosphorylation, mediated by the enhanced activity of eIF-4 alpha kinase(s) and inhibition of eIF-4 alpha phosphatase activity. In the resting cell, eIF-4 alpha is associated with heat- and acid-stable insulin-responsive protein (PHAS-I; 4E-BP1); following acute stimulation with phorbol ester, there is a 40% decrease in the amount of PHAS-I associated with eIF-4 alpha. Incubation of anti-PHAS-I immunoprecipitates with extracts containing activated or immunprecipitated mitogen-activated protein kinase resulted in a small increase in phosphorylation of recovered PHAS-I and a modest release of eIF-4 alpha from the PHAS-I-eIF-4 alpha complex. These data suggest a possible role for PHAS-I in the regulation of eIF-4F complex formation and the rate of translation in primary cells.
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton, U.K.
S J
SJ
Morley
V M
VM
Pain
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins; D003208:Concanavalin A / Q000494:pharmacology; D039562:Eukaryotic Initiation Factor-4F; D005786:Gene Expression Regulation / Q000187:drug effects; D007106:Immune Sera; D015151:Immunoblotting; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D013462:Sulfur Radioisotopes; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002352:Carrier Proteins; D039562:Eukaryotic Initiation Factor-4F; D007106:Immune Sera; D007166:Immunosuppressive Agents; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D010455:Peptides; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011090:Polyenes; D013462:Sulfur Radioisotopes; D003208:Concanavalin A; D008715:Methionine; D011494:Protein Kinases; C095649:eif-4alpha kinase; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate; D020123:Sirolimus