Project Summary/Abstract Previous work funded by this R35 grant led to the discovery that oncogenic viruses stimulate the production of a set of lipids that were not previously known to exist: phosphatidylinositol-3-phosphate, phosphatidylinositol- 3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate. This led to the discovery of the family of enzymes that generate these lipids, the phosphoinositide 3-kinases (encoded by the PIK3C family of genes). Subsequently, the PTEN tumor suppressor gene was shown to dephosphorylate the 3 position of these lipids. It is now clear that activating mutations in the PIK3CA gene and loss of function mutations in the PTEN gene are among the most common events driving the growth of cancers. Research funded by this grant revealed that the lipid products of PI3K directly activate a subset of protein Ser/Thr kinases, most notably the AKT family, and thereby initiate a set of protein kinase cascades, including activation of the mTOR protein kinase that coordinates a set of anabolic networks necessary for cell growth. Importantly, this research ultimately revealed that PI3Kalpha mediates the ability of insulin and IGF1, as well as other growth factors and oncogenic mutations, to stimulate glucose uptake into tissues. In order to identify the downstream protein substrates of AKT, mTOR and the host of other protein-Ser/Thr kinases and protein-Tyr kinases that regulate cell metabolism and cell growth, the funds from this grant were used to generate a set of oriented synthetic peptide mixtures, 10 residues in length, in which specific amino acids were locked-in at a specific position and all other positions had a mixture of all 20 amino acids. This allowed, for each human protein Ser/Thr or Tyr kinase, the identification of the optimal amino acid at each position, up to 5 residues N-terminal and up to 4 residues C-terminal of the Ser/Thr or Tyr that is phosphorylated. A manuscript describing the substrate specificity of all the Ser/Thr kinases was published in Nature this year and a paper describing the substrate specificity of all human Tyr kinases got a favorable review at Nature and is in revision. During the next granting period we will utilize the software described in the two Nature papers to determine what protein kinases are responsible for directly phosphorylating the hundreds of Ser, Thr and Tyr residues observed on enzymes that mediate anabolic metabolism. By comparing mass spec phosphoproteomics in cancers to normal tissues this approach will reveal the protein kinase cascades that drive anabolic metabolism in cancers and reveal new targets for pharmaceutical intervention to prevent survival of tumor cells.