Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.
In response to decreased cellular oxygen concentrations the basic helix-loop-helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia-inducible transcription factor, HIF-1alpha, mediates activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. Here we demonstrate that the mechanism of activation of HIF-1alpha is a multi-step process which includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation was shown to be dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1alpha was inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1alpha and recruitment of CBP. However, nuclear localization per se was not sufficient for transcriptional activation, since fusion of HIF-1alpha to the heterologous GAL4 DNA-binding domain generated a protein which showed constitutive nuclear localization but required hypoxic stimuli for function as a CBP-dependent transcription factor. Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and play critical roles in signal transduction by HIF-1alpha.
Kallio
P J
PJ
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Okamoto
K
K
O'Brien
S
S
Carrero
P
P
Makino
Y
Y
Tanaka
H
H
Poellinger
L
L
D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D015687:Cell Hypoxia; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D018257:Helix-Loop-Helix Motifs; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008164:Luminescent Proteins / Q000235:genetics; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D049452:Green Fluorescent Proteins