Acute myelogenous leukemia–derivedSMAD4 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.

TGF-β signaling in the control of hematopoietic stem cells

“…83 Nevertheless,a number of cases have been reported involving SMAD4 and TGFBR2 in patients with acute myelogenous leukemia (AML). [84][85][86][87] For example, in a study investigating human AML genomes, various copy number alterations were found recurrently modified, 1 of which represented the deletion of SMAD4.…”

The role of Smad signaling in hematopoiesis and translational hematology

“…[87][88][89][90] Using ultra-dense array comparative genomic hybridization on 86 AML genomes, 18 copy number alterations regions were found recurrently modified, one of which represented the deletion of the SMAD4 gene, demonstrating that SMAD4 can be an AMLassociated gene (Walter, PNAS, 2009). Furthermore, sporadic mutations in both TbRI and TbRII have been reported in lymphoid neoplasms.…”

MicroRNA-130a–mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors

“…15 A relation between aberrations in the TGF-␤-signaling pathway and acute myelogeneous leukemia (AML) has also been reported. [16][17][18] miRNAs are increasingly acknowledged as important regulators of translation and hence protein synthesis. miRNAs are Submitted March 11, 2011; accepted October 8, 2011.…”

“…15 A relation between aberrations in the TGF-␤-signaling pathway and acute myelogeneous leukemia (AML) has also been reported. [16][17][18] miRNAs are increasingly acknowledged as important regulators of translation and hence protein synthesis. miRNAs are 19-25 nucleotide noncoding RNA molecules that can regulate gene expression posttranscriptionally by binding to partially complementary sequences, mainly in the 3Ј-untranslated region (3Ј-UTR) of their target mRNA.…”