Growth Factor Receptor Binding Protein Bim-mediated Cell Apoptosis

Apoptosis is a normal physiological progress for killing unwanted cells in multicellular organisms. It is necessary in embryonic development and tissue homeostasis. The participation of mitochondria in apoptosis is one of the most important pathways. Mitochondrial pathway is regulated by proapoptotic and antiapoptotic Bcl-2 family proteins. BH3-only protein Bim was firstly discovered by an expression screen for proteins that bind to Bcl-2. Up to date, there are at least eleven isoforms of Bim have been reported. All of three major isoforms of Bim, Bim EL, L and S are able to bind to Bcl-2 and Bcl-x-L which reside on the mitochondrial outer membrane, and initiate cytochrome C release in certain settings of apoptosis. The apoptotic activity of Bim EL and Bim L is suppressed by binding to dynein motor complex via the Dynein light chain, LC8. Bim S lacks the binging domain to Dynein light chain and thus has the most potent cytotoxicity. The transcription of Bim gene can be regulated by transcription factors, such as FoxO3 (FKHRL1 ) ,Myb and RUNX3. JNK- mediated phosphorylating on certain amino acids of Bim can active Bim, and Erk1/2 can also promote Bim degradation through proteasome- dependent pathway. After Rb activated, E2F can bind to the promoter of Bim and increase the transcription of Bim. TGF-βcan up-regulate the transcription of Bim through TGF-β- Smads pathway and TGF-βcan also activative RUNX3 to promote Bim transcription. The interaction between Mcl-1 and Bim can inhibit the apoptosis induced by Bim. Bim can be disaggregated through the activated ERK1/2- mediated phosphorylation on the serine of Bim.To investigate the proteins interacting with Bim, we performed yeast two-hybrid using pLexA- Bim L as bait to screen human fetal brain library pB42AD. In the positive clones which repeat twice or more than twice, we focus the research on the growth factor receptor binding protein 10 (Grb10). Through the informatics and multiple tissues RT- PCR analysis, we found that Grb10 is ubiquitously expressed in multiple tissues especially in insulin target tissues such as pancreas and skeleton muscle, which suggesting the important function of Grb10 in insulin receptor pathway. Subcellular co-location of Bim L and Grb10 revealed that over-expression Bim L dispersed in the cytoplasm in short time and would diffuse to whole cells in long time. Grb10 mainly expressed surround the nuclear. We found that Grb10- RFP and mitochondria merged partly through dying by Mito- tracker. The result suggested that Grb10 might locate on the member of mitochondria and plasma. GFP- Bim L and Grb10- RFP merged partly suggested the interaction possibility between Grb10 and Bim. Through co-immunoprecipitation we confirmed the interaction between Grb10 and Bim. Moreover, Grb10 inhibited the apoptosis induced by over-expressed Bim L apparently by apoptosis assay. A lot of references have reported that SH2 domain of Grb10 can specifically interact with phosphorylated tyrosine receptor. Two tyrosine sites in Bim L were mutated. But Grb10 still inhibited the apoptosis induced by over-expressed mutated Bim L, which suggested that interaction between Grb10 and Bim L in some other way. Furthermore, we compared the apoptosis induced by isoforms of Bim L, Bim S, Bimα2 and Bimα3 separately when Grb10 over-expressed. Combining with the different domains from these four isoforms, we speculate that DBD domain might be the region of Bim L interacting with Grb10. By yeast two-hybrid we found that Grb10 might interact with the proteins which almost participating the proliferation promotion or apoptosis inhibiting, such as TCPC, PSMA and ProTα. TCTP can regulateTsc-Rheb pathway and promote cell proliferation and prevent cell apoptosis. PSMA participates the degradation of phosphorylated Bim. ProTαcan inhibit the apoptosis body formation and negatively regulate caspase- 9 and inhibit the apoptosis.The interaction between Grb10 and Bim L suggests that Grb10 might participate in the apoptosis signaling pathway besides the function in the insulin receptor pathway, and Grb10 might regulate Bim L directly. Our results may shed some light on the function of Grb10 and Bim L and lay a solid foundation for further apoptosis induced by Bim study.