FANCL Ubiquitinates Beta-Catenin and Enhances Its Nuclear Function

Abstract

1335 Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss. A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct). Disclosures: No relevant conflicts of interest to declare.

Deletion of the Fanconi Anemia C Gene in Mice Leads to Skeletal Anomalies and Defective Bone Mineralization and Microarchitecture

“…In addition, serum DKK1 levels were found to correlate with BMD parameters in patients with osteoporosis . Interestingly, we and others have reported dysregulated Wnt signaling in cells derived from FA‐mutant mice and from patients with FA . Moreover, we previously reported DKK1 overproduction in FA‐deficient cells and in sera from FA mouse models …”

“…For instance, binding of PPARγ to β‐catenin lead to rapid degradation of β‐catenin, which activate preadipocyte differentiation . FA patient‐derived cells were shown to have a dysregulated Wnt/β‐catenin signal shown by lack of β‐catenin activation and altered expression of Wnt/β‐catenin target genes . One of the Wnt/β‐catenin dysregulated genes found in FA is DKK1 .…”

Mutant calreticulin‐expressing cells induce monocyte hyperreactivity through a paracrine mechanism

“…Whole‐cell lysates were lysed in buffer containing 0.5% Triton X‐100, 120 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA, 1 mM Na 2 VO 4 , and 1:300 protease Inhibitor cocktail (P8340; Sigma‐Aldrich). Nuclear and cytoplasmic fractions were prepared essentially as described . 2D PAGE analysis of lysates was performed with Invitrogen's ZOOM system.…”

Research Progress of β-catenin Post-Translational Modification and its Function

“…Dao 等人 [30] [32] 研究发现, P300 可乙酰化 -catenin 的 Lys345 位点, 该残基的乙酰化增加了 -catenin 与 TCF4 的亲和力. Ge 等人 [33] 研究发现, PCAF(P300/CBP associated factor)可乙酰化-catenin…”