Oligomerization-induced modulation of TPR-MET tyrosine kinase activity.

Phosphorylation, although necessary, may not be sufficient to fully activate many receptor tyrosine kinases (RTKs). Oligomerization-induced conformational changes may be necessary to modulate the kinetic properties of RTKs and render them fully functional. To investigate this regulatory mechanism, recombinant TPR-MET, a functionally active oncoprotein derivative of the RTK c-MET, has been expressed and purified for quantitative enzymatic analysis. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil motif from the nuclear pore complex (TPR). cytoMET, the monomeric analog of TPR-MET, has also been expressed and purified for comparative enzymatic analysis. ATP and peptide substrates have been kinetically characterized for both TPR-MET and cytoMET. Significantly, phosphorylated TPR-MET has smaller Km values for ATP (Km,ATP) and peptide substrates (Km,peptide) and a larger kcat relative to phosphorylated cytoMET. This provides the first direct evidence that receptor oligomerization and not simply activation loop phosphorylation modulates RTK enzymatic activity. The ATP dissociation constants (Kd,ATP) for the two enzymes also displayed significant differences. In contrast, the KI values for the ATP competitive inhibitor staurosporin are similar for the two phosphorylated enzymes. These results suggest that much of the oligomerization-induced kinetic changes occur with respect to peptide substrate binding or catalytic efficiency. The possibility that oligomerization-induced conformational changes occur within the cytoplasmic domain of receptor tyrosine kinases has significant implications for structure-based design of RTK inhibitors and the development of a detailed mechanistic model of RTK activation.