Protein tyrosine kinases (PTKs) play pivotal roles in human cancer and are the targets of a major class of emerging anti-cancer drugs. In a single tumor, multiple PTKs are active and a substantial number are essential for maintaining the transformed phenotype. Though large numbers of phosphorylation sites have been mapped in cancer cells through mass spectrometry (MS), the identity of the specific kinases that phosphorylate these sites are with very few exceptions unknown. We propose to use emerging peptide microarray technology to identify consensus phosphorylation sequences for the entire set of human PTKs. We will generate a set of mammalian expression vectors producing every PTK fused to glutathione S-transferase. Each PTK will be affinity purified from a mammalian cell overexpression system and subjected to peptide microarray screening. These screens will reveal specific sequences preferred by each kinase at phosphorylation sites in their target substrates. We will use this data to mine phosphoproteomics data from cancer cells to connect known sites of phosphorylation to their respective kinases. Predicted kinase-substrate relationships will be validated through siRNA knockdown of the relevant kinase in cancer cell lines. The broad goal of this work is to elucidate critical connections in phosphorylation networks in cancer cells. These studies will enrich our understanding of the basic mechanisms of cellular transformation and tumor maintenance, provide insight into the mechanisms of action of kinase-targeted therapeutics, and suggest new targets for therapeutic intervention.

Public Health Relevance

Protein tyrosine kinases play pivotal roles in human cancer and are the targets of a major class of emerging anti-cancer drugs. This proposal will apply emerging peptide microarray technology to better understand how these kinases function in cancer by revealing how they interact with other molecules in cancer cells. This research will identify new molecules that cooperate with kinases in cancer and will suggest new targets for cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21CA147993-01
Application #
7852575
Study Section
Special Emphasis Panel (ZCA1-SRLB-V (J1))
Program Officer
Knowlton, John R
Project Start
2010-04-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
1
Fiscal Year
2010
Total Cost
$179,981
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520