DESCRIPTION: 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.
NIH ExPorter
NIH ExPorter