Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor B promoter/enhancer elements using tumor necrosis factor ␣, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.
Growth signals, such as extracellular nutrients and growth factors, have significant impacts on genome integrity, while the direct underlying link remains unclear. Here we show that the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) pathway, a central regulator of growth signaling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis, and impair its function in DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumor suppressor LKB1/STK11 hyper-activates the mTORC1-S6K signaling and decreases RNF168 expression, resulting in defects of DNA damage response. Expression of a phospho-deficient RNF168 (S60A) mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of the mTORC1-S6K signaling in DNA damage response and suggest a general mechanism connecting cell growth signaling to genome stability control.
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