Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1.
Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70(S6K1)-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.
Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Frankfurt, Germany.
Johanna S
JS
Blees
Heidi R
HR
Bokesch
Daniela
D
Rübsamen
Kathrin
K
Schulz
Larissa
L
Milke
Magdalena M
MM
Bajer
Kirk R
KR
Gustafson
Curtis J
CJ
Henrich
James B
JB
McMahon
Nancy H
NH
Colburn
Tobias
T
Schmid
Bernhard
B
Brüne
D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C471644:BTRC protein, human; C114487:PDCD4 protein, human; D016601:RNA-Binding Proteins; D012717:Sesquiterpenes; D044844:beta-Transducin Repeat-Containing Proteins; C063272:erioflorin acetate; D044767:Ubiquitin-Protein Ligases