N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H

J Cell Sci. 2010 May 1;123(Pt 9):1438-48. doi: 10.1242/jcs.067819. Epub 2010 Mar 31.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brefeldin A / pharmacology
  • COS Cells
  • Cell Line, Tumor
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism*
  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism
  • Chlorocebus aethiops
  • Cyclic AMP Response Element-Binding Protein / chemistry
  • Cyclic AMP Response Element-Binding Protein / genetics
  • Cyclic AMP Response Element-Binding Protein / metabolism*
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism
  • Glycosylation / drug effects
  • Humans
  • Mice
  • Mutant Proteins / metabolism
  • Proprotein Convertases / metabolism
  • Protein Binding / drug effects
  • Protein Folding / drug effects
  • Protein Processing, Post-Translational* / drug effects
  • Protein Stability / drug effects
  • Protein Structure, Quaternary
  • Protein Transport / drug effects
  • Serine Endopeptidases / metabolism
  • Transcription, Genetic / drug effects
  • Transcriptional Activation / drug effects

Substances

  • Creb3l3 protein, mouse
  • Cyclic AMP Response Element-Binding Protein
  • Mutant Proteins
  • Brefeldin A
  • Proprotein Convertases
  • Serine Endopeptidases
  • membrane-bound transcription factor peptidase, site 1