Although the tyrosine protein kinases (TPKs) are recognized as key enzymes in normal cellular growth and differentiation and mutated forms are linked to human disease, little is known about the structure of these catalysts or the mechanism of activity regulation. This paucity of structure-function information is due, in part, to the limited number of actively and solubly expressed TPKs. We have expressed and purified a soluble and active form of the kinase domain of the nonreceptor TPK, v-Fps, an oncoprotein linked to myocyte proliferation and congestive heart failure. Unlike the prototypical nonreceptor TPK, c-Src, the Src homology-2 (SH2) domain of v-Fps enhances, rather than represses, catalytic activity in the absence of receptor TPK binding. This mode of regulation makes v-Fps and its subfamily members unique and emphasizes the need for a thorough understanding of domain-domain interactions in these catalysts. We propose that the SH2 domain activates the kinase domain through specific domain-domain interactions that alter one or more active site residues. We present preliminary data (surface plasmon resonance and NMR techniques) that show that the isolated SH2 domain of v-Fps will only bind a phosphotyrosine peptide when the domain is conjugated to the putative A helix of the kinase domain may be an important region of the domain interface. We will express and purify an active, soluble SH2-kinase construct of v-Fps to determine the influence that the SH2 domain has on the catalytic mechanism using pre-steady-state kinetic and viscosometric methods. We will then make active-site mutations to determine the effects that this domain has on the role of these residues in catalysis. Secondary structural analyses will be performed on the extended SH2 domain using circular dichroism and multidimensional NMR for domain-domain interactions. We will determine whether this modification affects the structure of the SH2-kinase protein using size exclusion chromatography, proteolysis studies, optical spectroscopy nd site-directed mutagenesis. Having already overcome a major expression problem for this TPK, several new truncated forms of the kinase domain will be expressed to improve solubility. These new protein constructs will seed long-range goals which include the solution of X-ray structures for the kinase and SH2-kinase domains.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA075112-05
Application #
6173176
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Gallahan, Daniel L
Project Start
1997-08-20
Project End
2002-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
5
Fiscal Year
2000
Total Cost
$189,248
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Adams, Joseph A (2003) Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? Biochemistry 42:601-7
Hamuro, Yoshitomo; Wong, Lilly; Shaffer, Jennifer et al. (2002) Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS). J Mol Biol 323:871-81
Aubol, Brandon E; Nolen, Brad; Vu, Don et al. (2002) Mechanistic insights into Sky1p, a yeast homologue of the mammalian SR protein kinases. Biochemistry 41:10002-9
Shaffer, J; Sun, G; Adams, J A (2001) Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk. Biochemistry 40:11149-55
Leon, B C; Tsigelny, I; Adams, J A (2001) Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps. Biochemistry 40:10078-86
Hirai, T J; Tsigelny, I; Adams, J A (2000) Catalytic assessment of the glycine-rich loop of the v-Fps oncoprotein using site-directed mutagenesis. Biochemistry 39:13276-84
Konkol, L; Hirai, T J; Adams, J A (2000) Substrate specificity of the oncoprotein v-Fps: site-specific mutagenesis of the putative P+1 pocket. Biochemistry 39:255-62
Saylor, P; Hanna, E; Adams, J A (1998) Mutations in the activation loop tyrosine of the oncoprotein v-Fps. Biochemistry 37:17875-81
Saylor, P; Wang, C; Hirai, T J et al. (1998) A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps. Biochemistry 37:12624-30