Decreased ER-associated degradation of alpha-TCR induced by Grp78 depletion with the SubAB cytotoxin.
HeLa cells stably expressing the alpha chain of T-cell receptor (alphaTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2alpha phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of alphaTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of alphaTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of alphaTCR, confirming the role of VCP in ERAD of alphaTCR. It therefore appears that ERAD of alphaTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.
Lass
Agnieszka
A
Department of Anatomy and Cell Biology, Indiana University School of Medicine-Evansville, Evansville, IN 47712, USA.
Kujawa
Marek
M
McConnell
Elizabeth
E
Paton
Adrienne W
AW
Paton
James C
JC
Wójcik
Cezary
C
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins / Q000494:pharmacology*; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D034622:RNA Interference; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013381:Subtilisins / Q000494:pharmacology*
D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011948:Receptors, Antigen, T-Cell; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli