title: Acute myelogenous leukemia-derived SMAD4 mutations target the protein to ubiquitin-proteasome degradation.
abstract: Disruption of transforming growth factor-beta (TGFB1/TGF-beta) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-beta signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with protein instability; however, the precise mechanism by which mutated Smad4 proteins undergo rapid degradation remains to be elucidated. A missense mutation of the SMAD4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Delta483-552) have been identified in acute myelogenous leukemia. It is not known whether protein instability of these SMAD4 mutants is one of the contributors to TGF-beta signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia-derived SMAD4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that beta-transducin-repeat-containing protein 1 (beta-TrCP1), an F-box protein in the ubiquitin E3 ligase Skp1-Cullin-F-box protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated SMAD4 proteins. In addition, small interference RNA (siRNA)-triggered endogenous beta-TrCP1 suppression increased the protein expression level of both overexpressed SMAD4 mutants and endogenous mutated SMAD4 protein in acute myelogenous leukemia cells. These data suggest that mutated SMAD4 proteins undergo rapid degradation in acute myelogenous leukemia cells via SCF(beta-TrCP1) E3 ligase-mediated protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation.
authors.affiliation: School of Medicine, Shihezi University, Shihezi, Xinjiang, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.forename: Xu
authors.initials: X
authors.lastname: Cao
authors.forename: Mei
authors.initials: M
authors.lastname: Wan
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D016368:Frameshift Mutation; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D051901:Smad4 Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C471644:BTRC protein, human; D034741:RNA, Small Interfering; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; D044843:SKP Cullin F-Box Protein Ligases; D046988:Proteasome Endopeptidase Complex