Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia.
Phosphorylation-dependent ubiquitination combined with proteasomal degradation of transcriptional regulators is a recently appreciated mechanism for control of a number of inflammatory genes. Far less is known about the counterregulatory mechanisms that repress transcriptional activity in these pathways during resolution. Here, we investigated the transient nature of hypoxia-induced tumor necrosis factor (TNF)alpha in T84 cells, a process we have previously shown to involve phosphorylation-dependent degradation of the cAMP-response element-binding protein (CREB). Initial studies indicate hypoxia-induced TNFalpha to be a transient event, the resolution of which is associated with the appearance of a higher molecular weight modified form of CREB. Gene array analysis of mRNA derived from hypoxic cells identified a time-dependent induction of small ubiquitin-related modifier (SUMO)-1 mRNA. In prolonged hypoxia, CREB is posttranslationally modified by SUMO-1. Furthermore, SUMO-1 overexpression stabilizes CREB in hypoxia and enhances CREB-dependent reporter gene activity. Site-directed mutagenesis of lysine residues K285 and K304 identifies them as SUMO acceptors in vivo and in vitro. Mutation of K304 also results in loss of CREB nuclear localization, implying a role for SUMO-1 modification at this site in the subcellular localization of CREB. Thus, in prolonged hypoxia, CREB is modified by association with SUMO-1. Furthermore, we hypothesize that such an event stabilizes and promotes nuclear localization of CREB and thus complements an endogenous resolution phase for hypoxia-induced inflammatory processes.
Comerford
Katrina M
KM
The Conway Institute for Biomolecular and Biomedical Research, University College, Dublin, D4 Ireland.
Leonard
Martin O
MO
Karhausen
Jorn
J
Carey
Robyn
R
Colgan
Sean P
SP
Taylor
Cormac T
CT
D020816:Amino Acid Motifs; D000818:Animals; D019921:Biotinylation; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015696:Gene Products, tat / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025842:SUMO-1 Protein / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D015696:Gene Products, tat; D010455:Peptides; D012333:RNA, Messenger; D025842:SUMO-1 Protein; D014409:Tumor Necrosis Factor-alpha