Protein phosphorylation is considered the main regulatory switch of the cell, controlling processes such as cell growth, proliferation, differentiation and survival. This control Is performed by Intricate signaling cascades capable of changing proteins'activities and rapidly communicating messages from different extra cellular or internal cues to promote adequate cell readjustments. Overexpression, mutation or loss of some components in these networks, impede the proper functioning of this regulatory system, and constitute the hallmark of most human cancers. Cancer is considered to be a genetic disease: DNA mutations, copy number aberrations, chromosomal rearrangements and modifications in DNA methylation contribute in the development and progression of human tumors. Genomic analysis of these features has shown high diversity, in part due to the difficulties in discriminating mutations leading to cancer from those without functional effect. Most of this variation converges into aberrant protein phosphorylation patterns in a single or at most two signaling pathways simultaneously, thus studies of protein phosphorylation have the potential of revealing a common molecular signature in cancer from distinct mutation backgrounds. Novel mass spectrometry-based proteomics enables the analysis of protein phosphorylation en masse to offer a global picture of all phosphorylation events occurring in the cell. The goat of this proposal Is to approach the study of signaling pathways in cancer using state-of-the-art proteomics technology, with special emphasis In the Ras/MAPK and PI3K/Akt pathways and downstream effectors. This endeavor will be divided Into three specific alms: 1) Development of new technology for quantitative phosphoproteomlcs;2) Development and application of strategies for the identification substrates for basophilic kinases frequently upregulated in cancer of Akt;3) Characterization of deregulated signaling pathways in breast cancer cell lines.
Aims 1 and the methods part of Aim 2 have been completed during the mentored K99 phase of the grant.
Aims 2 and 3 are to be completed in the ROO phase, as the PI transitions to an Independent tenure-track academic position.
A broad characterization of signaling events in normal, klnase-actlvated and cancer cells will be invaluable to any researcher in cancer biology and translational research. Ras/MAPK and Akt/PI3K are of great Interest in drug discovery programs. Results derived from this research proposal will highly impact the design of efficient therapies.
|Eaton, James M; Takkellapati, Sankeerth; Lawrence, Robert T et al. (2014) Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation. J Biol Chem 289:18055-66|
|Edelman, William C; Haas, Kelsey M; Hsu, Joanne I et al. (2014) A practical recipe to survey phosphoproteomes. Methods Mol Biol 1156:389-405|
|Swaney, Danielle L; Beltrao, Pedro; Starita, Lea et al. (2013) Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nat Methods 10:676-82|
|Yu, Yonghao; Yoon, Sang-Oh; Poulogiannis, George et al. (2011) Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science 332:1322-6|