Cell-cell cohesion is a defining feature of multicellular organisms, and a number of disease states, from cancer to heart disease, are driven by defects in the structural "Velcro" that holds cells to one another. It is widely viewed that the cadherin/catenin adhesion complex is a master regulator of this adhesive "Velcro". This complex comprises a transmembrane "cadherin" component that mediates Ca++-dependent homophilic recognition, and a number of associated "catenins" that link cadherins to the underlying cytoskeleton. Since alpha-catenin (?-cat) is the sole actin-binding component of the cadherin/catenin complex, and filamentous (F)- actin is critical for strong intercellular adhesion, ?-cat has long been viewed as a key linker protein between cadherin adhesion receptors and the actin cytoskeleton. While loss of function studies show that ?-cat is essential for cell-cell adhesion across many cell types, the means through which this occurs is poorly understood. Our lab has discovered a highly conserved phosphorylation domain in ?-cat that is situated just N- terminal to a filamentous actin-binding site. These phosphorylations also lie within a mechano-sensitive, auto-inhibitory region of ?-cat, which restricts recruitment of another actin-binding protein to the cadherin/catenin complex via the central region of ?-cat. This proposal, therefore, seeks to understand how phosphorylation directs the conformational regulation, actin-binding and dynamic cell-cell adhesive functions of ?-cat using both in vitro and cell-based assays. Towards this end, Aim 1 seeks to show how in vitro phosphorylation of ?-cat at S641 by casein kinase 2 (CK2) enhances or "primes" for phosphorylation by CK1 at residues S652, S655 and T658, resulting in a more open conformation that favors ?-cat binding to F-actin and other actin-binding proteins.
Aim 2 will determine the contribution of these phosphorylations to cell adhesion in vivo, using a dog epithelial cell line that has replaced the endogenous ?-cat with GFP-tagged forms that contain, lack or constitutively mimic phosphorylation. Together, these aims address the fundamental question of how ?-cat interacts with actin at specialized adhesive junctions using both in vitro and cell-based approaches, which will have broad implications for intercellular adhesion across diverse cell types, and may be relevant to pathophysiological conditions that impact epithelial barrier function and overall tissue integrity.
Reduction in the levels of ?-cat has been observed in many different types of human cancers, and is a stronger prognostic factor for metastasis and patient survival than loss of E-cadherin, a bona fide tumor suppressor gene. In addition, deletion of ?-cat in mouse cardiomyocytes leads to severe disorganization of intercalated discs and the eventual development of dilated cardiomyopathy, suggesting that dysfunction of the cadherin/catenin complex could play a causal role in the development of cardiac disease. Thus understanding how ?-cat is regulated by phosphorylation will be broadly relevant to the study of diseases that impact tissue integrity, such as heart disease and cancer.
|Escobar, David J; Desai, Ridhdhi; Ishiyama, Noboru et al. (2015) Î±-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism. J Cell Sci 128:1150-65|
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