UC San Diego

The c-Abl kinase shuttles between the nucleus and cytoplasm. In the nucleus, Abl is activated by DNA damage or TNF to induce apoptosis. To examine the mechanism by which Abl regulates apoptosis, we employed dimerization as a means to control Abl activity by fusing an FKBP domain to the N-terminus of Abl. We found mutational inactivation of the NLS abrogated cell death, whereas FKBP-Abl was more efficient than FKBP-AblNuk, a nuclear-exclusive Abl, in inducing apoptosis, suggesting that while the nuclear pool is required for apoptosis, the cytoplasmic pool cooperates once apoptosis program is initiated. To find out how cytoplasmic Abl regulates cell adhesion, we made an AbPP gene that encodes an Abl protein that is constitutively activated. We stably transfected this gene into HEK293 cells under the control of a TET-on promoter. AblPP induction caused the host cells to detach from the supporting matrix in the presence of serum. We found that this was due to phosphorylation of CrkII by AblPP, leading to dissociation of the complex CrkII-C3G and loss of the GEF activity of C3G, resulting in the inactivation of Rap1 GTPase. The inactivation of Rap1 caused a decrease of integrin affinity for fibronectin. We also found that the Rho-ROCK1 pathway activated by LPA or serum is required for the cell detachment. These two pathways function independently of each other, and also play a role in ephrin A1-induced PC3 cell detachment. To further analyze the mechanisms of cell detachment induced by Abl, we employed shRNA library screening. We developed a new analytical strategy that combined quantitative microarray data with previous biological knowledge, i.e. Gene Ontology and pathway information, to increase the power of this technique. Using this strategy we identified 16 candidate shRNA-target genes associated with cell detachment induced by AblPP. Included in this set of genes was IL6ST, a membrane protein that we have experimentally confirmed. We also found that the caspase-resistant Rb-MI protein caused type-1 cells defect in Bid cleavage despite caspase-8 activation. Inhibition of V-ATPase, which is essential in acidification of endosomes, specifically restored Bid cleavage in Rb-MI cells, thus sensitizing Rb- MI but not wild-type fibroblasts to TNF-induced apoptosis, and stimulated inflammation-associated colonic apoptosis in RbMI/MI but not wild-type mice