GMCSF activates NF-kappaB via direct interaction of the GMCSF receptor with IkappaB kinase beta.
Granulocyte-macrophage colony-stimulating factor (GMCSF) has a central role in proliferation and differentiation of hematopoetic cells. Furthermore, it influences the proliferation and migration of endothelial cells. GMCSF elicits these functions by activating a receptor consisting of a ligand-specific alpha-chain and a beta-chain, which is common for GMCSF, interleukin-3 (IL-3), and IL-5. It is known that various signaling molecules such as Janus kinase 2 or transcription factors of the signal transducer and activator of transcription (STAT) family bind to the common beta-chain and initiate signaling cascades. However, alpha-chain-specific signal transduction adapters have to be postulated given that IL-3, IL-5, and GMCSF induce partly distinct biologic responses. Using a yeast 2-hybrid system, we identified the alpha-chain of the GMCSF receptor (GMRalpha) as putative interaction partner of IkappaB kinase beta, one of the central signaling kinases activating the transcription factor nuclear factor-kappaB (NF-kappaB). Using endogenous protein levels of endothelial cell extracts, we could verify the interaction by coimmunoprecipitation experiments. Fluorescence resonance energy transfer (FRET) microscopy confirmed the direct interaction of CFP-IKKbeta and YFPGMRalpha in living cells. Functional studies demonstrated GMCSF-dependent activation of IkappaB kinase activity in endothelial cells, degradation of IkappaB, and activation of NF-kappaB. Further biologic studies using GMCSF-dependent TF-1 cells indicated that GMCSF-triggered activation of NF-kappaB is important for cell survival and proliferation.
Ebner
Karin
K
Department of Vascular Biology and Thrombosis Research, University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria.
Bandion
Alexander
A
Binder
Bernd R
BR
de Martin
Rainer
R
Schmid
Johannes A
JA
D004730:Endothelium, Vascular / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D014471:Umbilical Veins / Q000166:cytology
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D021122:Protein Subunits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D000072000:NF-KappaB Inhibitor alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human