Functional Interaction Between HAX-1 And C-Abl Non-receptor Tyrosine Kinase

Mammalian non-receptor tyrosine kinase c-Abl is a modulatory protein with multiple biological functions. It plays important roles in the regulation of cell proliferation, apoptosis, adhesion, cell migration and stress responses. Employing full length c-Abl as bait protein for yeast two-hybridization, HS1 associated protein X-1 (HAX-1), which was involved in apoposis and cell mobility control by a not well addressed mechanism, was found potentially interact with c-Abl.In this study, interaction between HAX-1 and c-Abl was demonstrated by co-immunoprecipiation and GST pulldown assay. c-Abl was demonstrated to interact with HAX-1 through a domain covering 150-176aa by c-Abl SH3 domain. Tyrosine phosphorylation of c-Abl was found to be potentiated by bind to HAX-1 as confirmed by Westernblot with the anti-P-Tyr antibody, these results suggest that c-Abl be activated by HAX-1 by bind to its SH3 domain. Overexpression of HAX-1 down-regulates c-Abl protein level in a dose-dependent mannor. To further demonstrate the observation in physiological context, expression of HAX-1 in MCF-7 cells was knocked down by siRNA, and the expression of c-Abl was found to be up regulated in the cells depleted of HAX-1. In concert with the finding that the C terminal of HAX-1 interact with c-Abl, HAX-1 fragments containing 150-176aa, was able to down-regulate the c-Abl protein level. To reveal the mechanism, mRNA level in wild type and the HAX-1 deficient cells were accessed by RT-PCR, little if any effect was observed. It indicates that HAX-1 regulate c-Abl expression by a mechanism other than mRNA level. When the cells were treated with protein synthesis inhibitor CHX, it was found that c-Abl degraded much more fast when HAX-1 existed. It suggests that HAX-1 potentiate the degradation of c-Abl. Further, half lives of c-Abl in HEK293 cells transfected with or without HAX-1 were determined, and a shorter half live of c-Abl was observed in the cells overexpressing HAX-1 (3.31h) compared to the control (6.17h).It indicates that HAX-1 induces degradation of c-Abl though proteasome pathway.c-Abl plays important role in cell apoptosis through P73 pathway. To investigate whether HAX-1 has a role in c-Abl mediated cell apoptosis, MCF-7 and MCF-7/HAX-1siRNA cells were treated with apoptosis induce agents such as IR and cisplatin. More cell apoptosis was observed in the cells in which HAX-1 was knocked down. And the difference was decreased in the presence of the c-Abl kinase inhibitor, STI571. It suggest that the anti-apoptosis property is dependet on the presence of kinase active c-Abl.c-Abl is involved in the regulation of cellular reactive oxygen species by activating catalase and glutathione peroxidase 1 in physiological condition, and inactivate these two enzyme at higher ROS level by inducing ubiquitin modification and proteasomal degradation. We found in this study that HAX-1 is also involved in cell apoptosis induced by ROS. Cells resisted to H2O2 treatment in presence of HAX-1 were result from activated catalase and GPx1 with more active c-Abl which actived by HAX-1. It is also supported by the findings that intra cellular ROS is much higher in the HAX-1 knockdown cells compared to the wild type cells in the absence of c-Abl inhibitor STI571. Less difference was shown when the cells were treated with higher concentration of H₂O₂, since catalase and GPx1 is tend to be inactivated by c-Abl as well as the fact that c-Abl protein level is substantially decreased. Further, cellular ROS increases significantly in HAX-1 knockdown cells by the treatment of GPx1 inhibitor, it suggest that mitochonda GPx1 is more important than catalase in ROS metabolism regulated by HAX-1.These results provide a new insight in the regulation of c-Abl kinase, and may partially explain the anti-apoptotic role of HAX-1 in the cells.