Protein phosphorylation is the most common reversible post-translational modification in eukaryotes, yet signaling networks comprising protein kinases, their regulators, and their substrates are only partially elucidated. The overall goals of the proposed project are to build a comprehensive collection of consensus phosphorylation motifs for the entire collection of protein kinases encoded in the human genome using arrayed positional scanning peptide libraries, and integrate this data into web-accessible tools that are currently available to the entire biomedical community. The resulting dataset of protein kinase specificity motifs and informatics tools will: (1) allow functional annotation of a large number of proteins whose phosphorylation sites already have been, or currently are, being mapped in high-throughput phosphoproteomic mass- spectrometry experiments and datasets that have been previously funded by the NIH by now identifying the relevant kinase and signaling pathways responsible for these modifications;(2) allow the identification of new protein kinase substrates relevant to human health and disease and place them within the context of specific signal transduction pathways;and (3) provide a general set of kinase tools useful for structural and drug inhibitor studies and for the therapeutic targeting of specific signaling pathways implicated in human disease. To illustrate the utility of our approach, we will investigate predicted substrates of protein kinases in the Hippo signaling pathway, a conserved tumor suppressor pathway important in regulating cell proliferation, differentiation and survival. This project will serve to increase our fundamental understanding of how specificity is achieved by protein kinases, will identify critical connections in signaling networks, and will provide a general resource for researchers studying signal transduction and protein phosphorylation.

Public Health Relevance

Protein kinases play key roles in human disease and are important targets for some anti-cancer drugs. In this research, we are using high-throughput methods to analyze the function of every human kinase. This work will help in determining which kinases will be effective drug targets, and will provide resources to assist in kinase-targeted drug discovery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104047-02
Application #
8731945
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2013-09-10
Project End
2018-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$483,863
Indirect Cost
$98,549
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Cobbaut, Mathias; Derua, Rita; Döppler, Heike et al. (2017) Differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P+1 loop. Sci Rep 7:887
Goldberg, Allison B; Cho, Eunice; Miller, Chad J et al. (2017) Identification of a Substrate-selective Exosite within the Metalloproteinase Anthrax Lethal Factor. J Biol Chem 292:814-825
Mathea, Sebastian; Abdul Azeez, Kamal R; Salah, Eidarus et al. (2016) Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib. ACS Chem Biol 11:1595-602
Zhang, Yuan-Wei; Turk, Benjamin E; Rudnick, Gary (2016) Control of serotonin transporter phosphorylation by conformational state. Proc Natl Acad Sci U S A 113:E2776-83
Miller, Chad J; Turk, Benjamin E (2016) Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries. Methods Mol Biol 1360:203-16
Deng, Yang; Turk, Benjamin E (2016) Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays. Methods Mol Biol 1352:27-34
Goldberg, Allison B; Turk, Benjamin E (2016) Inhibitors of the Metalloproteinase Anthrax Lethal Factor. Curr Top Med Chem 16:2350-8
Cannell, Ian G; Merrick, Karl A; Morandell, Sandra et al. (2015) A Pleiotropic RNA-Binding Protein Controls Distinct Cell Cycle Checkpoints to Drive Resistance of p53-Defective Tumors to Chemotherapy. Cancer Cell 28:623-637
Egan, Daniel F; Chun, Matthew G H; Vamos, Mitchell et al. (2015) Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates. Mol Cell 59:285-97

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