Dao K.-H.T.,Oregon Health And Science University |
Rotelli M.D.,Oregon Health And Science University |
Brown B.R.,Oregon Health And Science University |
Yates J.E.,Oregon Health And Science University |
And 8 more authors.
Molecular Biology of the Cell | Year: 2013
Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48-linked chains. Evaluation of a series of N-terminal-deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3β is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3β. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function. © 2013 Dao et al.
Zhang Q.-S.,Oregon Health And Science University |
Marquez-Loza L.,Oregon Health And Science University |
Eaton L.,Oregon Health And Science University |
Duncan A.W.,Oregon Health And Science University |
And 10 more authors.
Blood | Year: 2010
Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2-/- mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit +Sca-1+Lineage- (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2-/- KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2-/- mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2-/- KSL cells in quiescence, improved the marrow micro-environment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2-/- bone marrow cells. We conclude that Fancd2-/- mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies. © 2010 by The American Society of Hematology.