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.