N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H.
CREB-H is a liver-enriched bZIP transcription factor of the CREB3 subfamily. CREB-H is activated by intramembrane proteolysis that removes a C-terminal transmembrane domain. Aberrant expression of CREB-H is implicated in liver cancer. In this study we characterized N-linked glycosylation of CREB-H in the luminal domain at the C-terminus. We found that CREB-H is modified at three N-linked glycosylation sites in this region. Disruption of all three sites by site-directed mutagenesis completely abrogated N-linked glycosylation of CREB-H. The unglycosylated mutant of CREB-H was not unstable, unfolded or aggregated. Upon stimulation with an activator of intramembrane proteolysis such as brefeldin A and KDEL-tailed site 1 protease, unglycosylated or deglycosylated CREB-H was largely uncleaved, retained in an inactive form in the endoplasmic reticulum, and less capable of activating transcription driven by unfolded protein response element or C-reactive protein promoter. Taken together, our findings suggest that N-linked glycosylation is required for full activation of CREB-H through intramembrane proteolysis. Our work also reveals a novel mechanism for the regulation of CREB-H-dependent transcription.
Chan
Chi-Ping
CP
Department of Biochemistry, The University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong.
Mak
To-Yuen
TY
Chin
King-Tung
KT
Ng
Irene Oi-Lin
IO
Jin
Dong-Yan
DY
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D043484:Proprotein Convertases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000187:drug effects; D012697:Serine Endopeptidases / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects
C500051:Creb3l3 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D050505:Mutant Proteins; D020126:Brefeldin A; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C115575:membrane-bound transcription factor peptidase, site 1