Cellular transformation and tumor formation by Rous sarcoma virus is dependent on the expression of the viral v-src gene. The product of this gene is a 60-kilodalton tyrosine protein kinase designated as v-Src. Likewise, the transforming gene of Abelson murine leukemia virus (which causes B-cell lymphomas in vivo) encodes a tyrosine protein kinase designated as v-Abl. This proposal focuses on the role of protein-protein interactions in the function of the v-Src and v-Abl tyrosine kinases. It has been suggested that the Src homology (SH) regions 2 and 3 of nonreceptor tyrosine kinases are important both in the regulation of kinase activity and in the recognition of cellular substrates. Regions of v-Src and v-Abl which are involved in intra- or intermolecular recognition will be identified by peptide-based photoaffinity labelling experiments. Peptide substrate analogs for these enzymes containing the photoactive amino acid p-benzoyl-Phe will be used as the affinity labels. Modified regions inside and outside the SH2 and SH3 domains will be studied further by site-directed mutagenesis, and the mutant enzymes will be tested by in vitro phosphorylation with tyrosine-containing synthetic peptides. These results will be correlated with a model of the three-dimensional structure of v-Src based on the recent crystal structure of the catalytic domain of the cAMP-dependent protein kinase. To identify determinants in protein substrates for v-Src which confer recognition by the wild-type and mutant enzymes, synthetic peptide """"""""libraries"""""""" will be used to select those peptides which give maximal phosphorylation by v-Src. Mutant v-Src kinases will be used to study in vivo substrate recognition. The model system for these studies will be Rat-1 cells transfected with v-Src constructs encoding kinases with altered specificity. Substrate recognition by the mutant enzymes will be assessed by three criteria: effects on total tyrosine phosphorylation, effects on phosphorylation of phospholipase C-gamma and other specific substrates, and effects on cellular transformation. The goal of these studies is to describe at a molecular level the steps leading to the formation of the """"""""signalling complexes"""""""" which play a central role in signal transduction and oncogenic transformation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA058530-05
Application #
2517577
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Cole, John S
Project Start
1993-09-01
Project End
1999-04-30
Budget Start
1997-09-01
Budget End
1999-04-30
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Physiology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Touchette, Megan H; Bommineni, Gopal R; Delle Bovi, Richard J et al. (2015) Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl ?-Diol Lipids. Biochemistry 54:5457-68
Tsui, Tiffany; Miller, W Todd (2015) Cancer-Associated Mutations in Breast Tumor Kinase/PTK6 Differentially Affect Enzyme Activity and Substrate Recognition. Biochemistry 54:3173-82
Krishnan, Harini; Ochoa-Alvarez, Jhon A; Shen, Yongquan et al. (2013) Serines in the intracellular tail of podoplanin (PDPN) regulate cell motility. J Biol Chem 288:12215-21
Schultheiss, Kira P; Craddock, Barbara P; Tong, Michael et al. (2013) Metazoan-like signaling in a unicellular receptor tyrosine kinase. BMC Biochem 14:4
Schultheiss, Kira P; Suga, Hiroshi; Ruiz-Trillo, IƱaki et al. (2012) Lack of Csk-mediated negative regulation in a unicellular SRC kinase. Biochemistry 51:8267-77
Yadav, Shalini S; Yeh, Brian J; Craddock, Barbara P et al. (2009) Reengineering the signaling properties of a Src family kinase. Biochemistry 48:10956-62
Patwardhan, Parag; Shiba, Kiyotaka; Gordon, Chris et al. (2009) Synthesis of functional signaling domains by combinatorial polymerization of phosphorylation motifs. ACS Chem Biol 4:751-8
Li, Wanqing; Scarlata, Suzanne; Miller, W Todd (2009) Evidence for convergent evolution in the signaling properties of a choanoflagellate tyrosine kinase. Biochemistry 48:5180-6