Functional Interaction Between C-Abl Non-receptor Tyrosine Kinase And FHL2

The c-abl gene was firstly identified as the cellular homologue of the transforming gene of Abelson murine leukemia virus. c-Abl is a non-receptor tyrosine kinase located both in the nucleus and the cytoplasm. c-Abl is involved in many cellular processes such as cell proliferation , differentiation, adhesion , migration,apoptosis and cell cycle by regulating various signaling pathways .c-Abl could be activated by growth factors such as EGF and PDGF, and is an effector of Src for growth factor induced c-myc expression and DNA synthesis. c-Abl is activated in response to ionizing radiation and regulates the cell cycle and cell apoptosis, but the mechanisms and the downstream effectors of c-Abl are not clearly understood. It was shown that FHL2 may be one of the potential c-Abl interaction proteins by the yeast two-hybrid experiment using full length c-Abl as bait protein in our previous work . FHL2 ,which is expressed mainly in heart and overexpressed in many cancer cell, is a transcription cofactor involved in cell proliferation and differentiation by regulating AP-1,Wnt/catenin and Rho signal pathway. However, the mechanisms responsible for the regulation of FHL2 in cells are not well known.In this study, direct interaction between FHL2 and c-Abl was demonstrated by co-immunoprecipiation, in vitro binding and GST-pull down assay. FHL2 binds to c-Abl through its motif containing P110 and P143 amino acids.FHL2 was shown to be the phosphorylation substrate of c-Abl by kinase assay, the Tyr93 and Tyr97 were the major tyrosine phosphorylation sites. c-Abl mediated phosphorylation of FHL2 resulted in self-assembly and nuclear translocation and thereby enhance the transcription activity of FHL2. The growth factor EGF, which is known to activate c-Abl ,could stimulate the transcription activity of FHL2 in MCF-7 dependent on kinase activity of c-Abl.. c-Abl was demonstrated to stimulate the activity of Wnt/β-catenin signal pathway by interacting with FHL2 . These results suggest that FHL2 is the downstream effector of c-Abl mediated the EGF and Wnt/β-catenin signal pathway.c-Abl is activated by ionizing radiation and FHL2 expression is also stimulated by ionizing radiation. In this study, we found that c-Abl and FHL2 are both involved in 4 ionizing radiation mediated G2/M cell cycle arrest. The c-Abl knockdown MCF-7 cells and FHL2 knockdown MCF-7 cells displayed defective G2/M arrest following exposure to ionizing radiation. Further ,we found that c-Abl could bind with Cdc25C and inhibit the Cdc25C phosphatase activity, but not Chk1 activity. Inhibition of Cdc25C phosphatase activity by c-Abl resulted in the accumulation of the Tyr15 phosphorylated Cdc2 following exposure to ionizing radiation and cell cycle progression from G2/M to G1 was impaired. FHL2 played roles in G2/M arrest mediated by ionizing radiation possibly by regulating steady level of the c-Abl protein ,since FHL2 could promote the stability of c-Abl protein through blocking its ubiquitination and proteasomal degradation .In summary, the study demonstrated that c-Abl associate with and phosphorylate FHL2 ,the interaction of c-Abl with FHL2 played important roles in cell proliferation, and particularly , G2/M cell cycle arrest after ionizing radiation.