Abstract 1457: Cbl, E3 Ubiquitin Ligase, is a Regulator of Focal Adhesion Protein Proteasomal Degradation during Cardiomyocyte Anoikis
Proteasome degradation of ubiquitin-targeted proteins is an important mechanism that negatively controls activated signaling pathways. The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor tyrosine kinase signaling, resulting in their ubiquitination and down-regulation. We have shown previously that neutrophil derived protease cathepsin G (Cat.G) induced focal adhesion (FA) protein degradation and myocyte apoptosis by anoikis. We hypothesized that Cbl is involved in myocyte apoptosis in response to Cat.G through ubiquitination and downregulation of FA proteins.
Results: Immunoprecitpitation and western blot studies showed increased Cbl tyrosine phosphorylation in response to Cat.G that was associated with its interaction with FA proteins, FAK and paxillin. This led to an increase in FAK and Paxillin ubiquitination and degradation at 2 hrs after Cat.G addition. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced FAK and paxillin degradation. In contrast, adenoviral expression of wild type Cbl enhanced FAK and paxillin ubiquitination and degradation in response to Cat.G. Interestingly, Cbl activation was involved in Cat.G-induced myocyte apoptosis as overexpression of dominant negative Cbl significantly reduced caspase-3 activation and DNA fragmentation induced by Cat.G. Concomitant with these changes in vitro, heart tissue samples from patients with ischemic or dilated cardiomyopathy showed a marked increase in Cbl accumulation and FAK and paxillin degradation compared to heart controls.
Conclusion: These studies show that Cbl abundance is increased in human ischemic and dilated cardiomyopathy and that activation of Cbl increases FA protein degradation and myocyte apoptosis. These results indicate that Cbl is a positive regulator of FA protein degradation and myocyte apoptosis and may play an important role in mediating the effect of neutrophil derived proteases during the progression of congestive heart failure.