We propose to apply the new technique of pulsed SILAC (pulsed stable isotope labeling by amino acids in cell culture) to generate and make freely available a global database of the effects of TOR inhibition on de novo protein synthesis. Pulsed SILAC determines the levels of proteins that are newly synthesized during a defined time interval and is fundamentally different from conventional SILAC which measures steady-state protein levels. We will determine de novo synthesis for several thousand proteins in the presence and absence of rapamycin and of the ATP-competitive TOR inhibitor PP242. The cells to be used in this study will be MCF10A, a human breast epithelial cell line, and MCF10A H1047R, the same cell line with a knock-in of the H1047R mutation of phosphoinositide 3-kinase (PI3K). In MCF10A cells, TOR is not stimulated in the absence of growth factors;in MCF10A H1047R cells, TOR activity is constitutively upregulated by elevated signaling from PI3K. The work will be carried out in collaboration with the laboratory of Professor John Yates at the Scripps Research Institute. Pulsed SILAC provides extensive coverage of the proteome. The proposed database will become a valuable resource and will greatly accelerate research on aging and on cancer.
We propose to generate a freely accessible database of changes in protein synthesis induced by the inhibition of the TOR kinase. The work will examine thousands of proteins and will create a valuable basic resource that will facilitate and accelerate research on aging and on cancer.