Molecular Mechanism Of Focal Adhesion Kinase Modulating The Activation Of The Mammalian Target Of Rapamycin Signaling Pathway Via Tuberin

mTOR, the mammalian target of rapamycin, which is a evolutionary conservative Ser/Thr kinase, belongs to the PIKK (phosphatidylinositol kinase-related kinase) super family. mTOR can integrate signals from growth factors, nutrition, energy and regulate cell proliferation and growth through it downstream effectors.Focal adhesion kinase (FAK) is a cytoplasm non-receptor protein tyrosine kinase. Through integrating signals from integrin, growth factors and stress, it regulates cell growth and migration. FAK serves as a centre for both inter and intracellular signal transduction.Tuberous Sclerosis Complex (TSC) is a kind of autosomal dominant disease caused by the mutation of TSC1or TSC2. Tuberin, encodes by TSC2, forms heterodimer together with Harmatin which is encoded by TSC1. The TSC2/TSC1complex serves as a negative upstream regulator of mTOR. It is reported that both FAK and Tuberin are the key regulating molecules of mTOR signaling pathway. However, the association of FAK with Tuberin is still illusive. Whether FAK regulates mTOR through directly interaction with Tuberin has not been well determined.We studied the mechanism of FAK and Tuberin in different cell lines, including293T (human), STO (mouse) and GFb (goat). At first, we elucidated the effect of FAK on regulating mTOR signaling pathway by RNAi. The results showed that the interference of FAK reduced the expression and phosphorylation level of mTOR pathway components, including Akt, mTOR, S6K and S6. Then we performed co-immunoprecipitaion with anti-FAK antibody and found that Tuberin interacted with FAK in vivo. Finally, we carried out yeast two-hybrid to study the specific domain by which FAK interacted with Tuberin.The results indicated that after FAK gene silencing, the activation of phospho-mTOR(Ser2448), phosphor-p70S6K(Thr389) and phospho-S6(Ser240/244) was inhibited, while the phosphorylation activity of phospho-Akt(Ser473) did not change. Further more, Tuberin was co-immunoprecipitated with FAK and Akt was not detected in co-immunoprecipitation complex. Besides, yeast two hybrid results showed that both full length FAK (aa.1-1052) and its C-terminal fragment named as FAK3(aa.665-1052), which including FAT domain, could interact with the middle fragment (aa.419-1080, TSC2F) of TSC2. FAK N-terminal FERM domain named as FAK1(aa.1-400) and the kinase domain named as FAK2(aa.401-664) have no or only weak interactions with the TSC2F. These results indicated that the FAK3 fragment is essential for its association with Tuberin.Taken together, combining and comparing of data obtained from experiments in different cell lines, it is believed that FAK interacts with Tuberin and this interaction depends on the C-terminal fragment of FAK included FAT domain which named FAK3. FAK can regulate the activation of mTOR signaling pathway through directly interaction with Tuberin. The molecular mechanism of crosstalk between FAK and TSC2/mTOR signaling pathway is highlighted.