title: TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation.
abstract: CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G2/M-checkpoint arrest upon H2O2 treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.
authors.affiliation: Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Nilay Y
authors.initials: NY
authors.lastname: Thakar
authors.affiliation: Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Ovchinnikov
authors.affiliation: Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, QLD 4029, Australia.
authors.forename: Marcus L
authors.initials: ML
authors.lastname: Hastie
authors.affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre and Institute for Molecular Bioscience, University of Queensland, St. Lucia, 4067 QLD, Australia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
authors.affiliation: Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, QLD 4029, Australia.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gorman
authors.affiliation: Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Ernst J
authors.initials: EJ
authors.lastname: Wolvetang
mesh_terms: D019943:Amino Acid Substitution; D017209:Apoptosis / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D002470:Cell Survival; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D017730:Ki-1 Antigen / Q000235:genetics* / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism*
chemical_list: D017730:Ki-1 Antigen; D016328:NF-kappa B; D047992:TNF Receptor-Associated Factor 2; D048049:Extracellular Signal-Regulated MAP Kinases