Identification Of Theinteraction Domain Of FAK And TSC2 By Yeast Two-Hybrid System And A TSC2 Phosphorylation Site By Mass Spectrometry

Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor protein-tyrosine kinase, which identified as a key mediator of integrins signaling pathways. FAK is overexpressed in cancer cells and related with adhesion, migration and invasion of tumor cells. TSC1and TSC2 are tumour suppressor genes mutated in the tumor syndrome tuberous sclerosis complex (TSC), which gene products (hamartin and tuberin) form a stable protein complex (TSC 1/2). The TSC 1/2 complex has been shown to modulate mammalian target of rapamycin (mTOR) complex1 (mTORC1) signaling pathways to regulate cell growth. mTOR is a Ser/Thr kinase, which is a key modulator of anabolic processes, exists in two major multi-protein complexes termed as mTORC1 and mTORC2.mTOR promotes protein synthesis, cell growth and cell proliferation in response to growth factors, energy levles, cellular stress and amino acids.Our previous study showed that FAK might interacted with TSC2 to regulate the mTOR signaling pathway. In order to further investigatethe interaction mechanism ofFAK and TSC2, we did the following experiments.1.The interaction domain of FAK and TSC2 was identified by yeast two-hybrid.2. The activity of mTOR, S6 and 4EBP1 were examined by western blot in human 293 T cells transfected by FAK overexpression vector and FAK RNA interference vector to confirm the regulatory role of FAK in mTOR signaling pathway.3. The interaction of FAK and TSC2 was examined by co-immunoprecipitaionin yeast with co-expression of FAK and TSC2. 4. The phosphorylation site of TSCC2phosphorylated by interacting FAK was examined by co-immunoprecipitaion and mass spectrometry in yeast with co-expression of FAK and TSC2, in which TSC2 is over-expressed after galactose induction. Ourexperiment results suggested the FAT domain(aa.665-1052) within C-terminal of FAK could interact with the hamartin domain(aa.1-419) within N-terminal fragment of TSC2, which results demonstrated that C-terminal fragment of FAK is essential for its interact with the hamartin of TSC2. The phosphorylation of FAK(Tyr397), mTOR(Ser2448),4EBP1(Thr37/46) and S6(Ser240/244) were enhanced in 2393 T cells transfected with FAK over expression vector, while the activities of these four molecules were inhibitied in 293 T cell transfected with FAK RNA interference vector. The co-immunoprecipitation result showed that FAK is interacted with TSC2 in yeast with co-expressed FAK and TSC2. The mass spectrometry results showed that TSC2 was phosphorylated at the site of Thr-779 by interacting FAKIn conclusion, our results indictes that FAT domain within C-terminal fragment of FAK interact with hamartin domain within N-terminal fragment of TSC2. FAK may phosphorylate the Tyr-779 site of TSC2 to regulate the mTORC1 signaling pathways.