Regulation of FANCL by Glycogen Synthase Kinase-3beta Links the Fanconi anemia pathway to Self Renewal and Survival Signals

Abstract 1263 The molecular basis for how a Fanconi anemia (FA) genetic background contributes to hematopoietic stem cell defects and hypoplastic organ development remains poorly understood. Protein modification by ubiquitination is a mechanism that diversifies the function and regulation of proteins. In light of this, we focus on the dysfunction of FANCL, the E3 ubiquitin ligase of the FA pathway, as a key molecular defect in Fanconi anemia. Here we report our studies investigating mechanisms of post-translational regulation of FANCL. We view these mechanisms as potential targets to augment the function of the FA core complex and correct hematopoietic stem cell defects. We provide evidence that FANCL is exquisitely regulated by ubiquitin-proteosome degradation. Ligase-inactive mutants (FANCL-C307A and -W341G) are less sensitive to this regulation, suggesting a role for auto-ubiquitination in directing lysine-48 polyubiquitination. This constitutive negative regulation of FANCL is partially reversed with an ATP-competitive glycogen synthase kinase-3beta (GSK-3beta) inhibitor. GSK-3beta is a serine/threonine kinase that phosphorylates proteins and marks them for ubiquitin-mediated proteolysis. Mitogenic and survival pathways, including Ras/MAPK and PI3K/Akt, negatively regulate GSK-3beta by serine-9 phosphorylation. We show that the regulation of FANCL by GSK-3beta is likely direct because FANCL and GSK-3beta co-immunoprecipitate in cell lysates and as GST-fusion proteins. To define the biochemical mechanisms of FANCL regulation, we generated N-terminal deletion mutants of FANCL and we show that the regulation of FANCL is dictated by a region at the N-terminus (aa1-78). Mutational analysis of FANCL (lysine to arginine) in this N-terminus region does not affect the overall protein level or ubiquitination of FANCL, suggesting that FANCL may be targeted for degradation by phosphorylation and/or in a complex with other proteins. The potential biological relevance of our findings, that FANCL is regulated by GSK-3beta is revealed in studies overexpressing constitutively active, myristoylated-Akt. This experimental condition increases FANCL protein levels and suggests a role for FANCL as a downstream effector of PI3K/Akt signaling. In turn, FANCL likely regulates non-canonical targets that alter the transcriptome profile favoring self-renewal and survival of hematopoietic stem cells. We recently published our studies identifying beta-catenin as one such downstream target (Blood 2012 Jul 12;120:323). Suppression of FANCL expression severely disrupts Wnt/beta-catenin signaling and expression of downstream Wnt-responsive targets MYC and CCND1. We also identified that GSK3B gene expression is approximately 5-fold higher in Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared to untreated cells or to wildtype cells with or without TNF-alpha. Our current studies show that inhibition of GSK-3beta preserves the number of murine Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared with no GSK-3beta inhibition. Taken together, we have accumulated evidence suggesting that GSK-3beta is a promising molecular target to improve the self-renewal and survival of FA hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.