The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex.
The eukaryotic translation initiation factor (eIF) 4E, is regulated by modulating both its phosphorylation and its availability to interact with the scaffold protein, eIF4G, to form the mature eIF4F complex. Here we show that treatment of C2C12 myoblasts with the proteasomal inhibitor, MG132 (N-carbobenzoxyl-Leu-Leu-leucinal), resulted in an early decrease in protein synthesis rates followed by a partial recovery, reflecting the reprogramming of translation. The early inhibition of protein synthesis was preceded by a transient increase in eIF2alpha phosphorylation, followed by a sustained increase in eIF4E phosphorylation. Inhibition of eIF4E phosphorylation with CGP57380 failed to prevent translational reprogramming or the moderate decrease in eIF4F complexes at later times. Prolonged incubation with MG132 resulted in the increased expression of heat shock protein (hsp)25, alphaB-crystallin and hsp70, with a population of hsp25 associating with the eIF4F complex in a p38 mitogen-activated protein kinase-dependent manner. Under these conditions, eIF4GI, and to a lesser extent eIF4E, re-localized from a predominantly cytoplasmic distribution to a more perinuclear and granular staining. Although MG132 had little effect on the colocalization of eIF4E and eIF4GI, it promoted the SB203580-sensitive association of eIF4GI and hsp25, an effect not observed with alphaB-crystallin. Addition of recombinant hsp25 to an in vitro translation assay resulted in stimulation of on-going translation and a moderate decrease in de novo translation, indicating that this modified eIF4F complex containing hsp25 has a role to play in recovery of mRNA translation following cellular stress.
Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton, UK.
Joanne L
JL
Cowan
Simon J
SJ
Morley
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003459:Crystallins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism*; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018832:Molecular Chaperones; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D012315:RNA Caps; D015398:Signal Transduction / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases
D003459:Crystallins; D015853:Cysteine Proteinase Inhibitors; D039562:Eukaryotic Initiation Factor-4F; D006360:Heat-Shock Proteins; C486113:Hsbp1 protein, mouse; D007976:Leupeptins; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D012315:RNA Caps; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde