Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia.
Hypoxia activates a number of gene products through degradation of the transcriptional coactivator cAMP response element binding protein (CREB). Other transcriptional regulators (e.g., beta-catenin and NF-kappa B) are controlled through phosphorylation-targeted proteasomal degradation, and thus, we hypothesized a similar degradative pathway for CREB. Differential display analysis of mRNA derived from hypoxic epithelia revealed a specific and time-dependent repression of protein phosphatase 1 (PP1), a serine phosphatase important in CREB dephosphorylation. Subsequent studies identified a previously unappreciated proteasomal-targeting motif within the primary structure of CREB (DSVTDS), which functions as a substrate for PP1. Ambient hypoxia resulted in temporally sequential CREB serine phosphorylation, ubiquitination, and degradation (in vitro and in vivo). HIV-tat peptide-facilitated loading of intact epithelia with phosphopeptides corresponding to this proteasome targeting motif resulted in inhibition of CREB ubiquitination. Further studies revealed that PP1 inhibitors mimicked hypoxia-induced gene expression, whereas proteasome inhibitors reversed the hypoxic phenotype. Thus, hypoxia establishes conditions that target CREB to proteasomal degradation. These studies may provide unique insight into a general mechanism of transcriptional regulation by hypoxia.
Taylor
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CT
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Furuta
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Synnestvedt
K
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Colgan
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SP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D023421:Models, Animal; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D014158:Transcription, Genetic; D014452:Ubiquitins / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D009097:Multienzyme Complexes; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex