Continued support is requested for our investigation of the roles of filamin in cell differentiation, invasion and disease. Filamins are essential actin crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like domains which interact with numerous cytosolic signaling proteins and transmembrane receptors. Humans have three filamin genes, encoding the widely expressed filamin A and B and largely muscle specific filamin C. Missense point mutations in filamins cause a variety of human diseases, ranging from altered neuronal migration, to cardiac and skeletal muscle defects, and a spectrum of congenital malformations generally characterized by skeletal dysplasias but also including extra-skeletal malformations such as cleft palate, cardiac defects and obstructive uropathy. The actin-binding domain is a hotspot for filamin mutations but, despite dramatic progress in understanding filamin structure and function, how filamin point mutations cause disease remains poorly understood. Furthermore, reduced filamin A expression correlates with increased breast cancer invasion and metastasis, and we recently discovered that loss of filamin increases extracellular matrix (ECM) remodeling and cell invasion. How filamin controls ECM degradation and invasion is unknown. In addition, we have shown that ASB2 (ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2), part of an E3 ubiquitin ligase complex, targets filamins for rapid proteasomal degradation and we suggest that the resultant transient loss of filamin contributes to retinoic acid-induced differentiation of acute promyelocytic leukemia cells. However, the molecular basis for ASB2 function and how loss of filamin influences cell differentiation have not been elaborated. To address the important unanswered questions highlighted above we propose three specific aims which draw on our extensive experience using biochemical, cellular and structural techniques to investigate filamins. Specifically, we will: 1) Characterize the mechanism of ASB2-mediated filamin-degradation and test its role in cell differentiation;2) Assess the role of filamins in EC remodeling and cell invasion;and 3) Identify cellular phenotypes associated with disease-associated filamin point mutations, revealing potential molecular mechanisms of disease.
Mutations in filamins, an important family of actin-bundling proteins, give rise to a range of congenital disorders including malformations of the brain, skeleton and cardiovascular system, and loss of filamin is correlated with increased breast cancer invasion and metastasis. We will use biochemical, cell biological and structural approaches to characterize the role of filamins in cell differentiation, invasion and extracellular matrix remodeling, and to identify the cellular phenotypes underlying diseases associated with filamin point mutations.
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