Striking multiplicity of eIF4E-BP1 phosphorylated isoforms identified by 2D gel electrophoresis regulation by heat shock.
Eukaryotic initiation factor eIF4E-binding protein 1 (eIF4E-BP1), or PHAS-I, is multiply phosphorylated by insulin-stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E-BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E-BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8-10 prominent eIF4E-BP1 isoforms were detected. Following heat shock, a rapid, temperature-dependent dephosphorylation of eIF4E-BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E-BP1 parallels restoration of protein synthesis. However, eIF4E-BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E-BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E-BP1. The amount of eIF4E-BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E-BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E-BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E-BP1)-specific antibodies. These results identify a complex set of eIF4E-BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.
Duncan
R F
RF
School of Pharmacy, Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA, USA. rduncan@hsc.usc.edu
Song
H J
HJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004330:Drosophila; D029721:Drosophila Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D019319:Okadaic Acid / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; C087000:EIF4EBP1 protein, human; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D047908:Intracellular Signaling Peptides and Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D020033:Protein Isoforms; D011994:Recombinant Proteins; C410006:Thor protein, Drosophila; D019319:Okadaic Acid; D017346:Protein Serine-Threonine Kinases