Oligomerization-induced Modulation of TPR-MET Tyrosine Kinase Activity

John L Hays,Stanley J Watowich

doi:10.1074/jbc.m210648200

Abstract

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.

Highlights

Among other cellular responses [1]
To investigate this regulatory mechanism, recombinant TPRMET, a functionally active oncoprotein derivative of the receptor tyrosine kinases (RTKs) 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 been expressed and purified for comparative enzymatic analysis
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

Summary

EXPERIMENTAL PROCEDURES

Materials and Reagents—All restriction endonucleases were from New England BioLabs, Taq pfu was from Promega, the Bac-to-Bac baculovirus expression system, SF-900II serum-free medium, fetal bovine serum, and 100ϫ antibiotic/antimycotic were purchased from. MANT-AMPPnP (a fluorescent non-hydrolyzable derivative of ATP) was synthesized and purified by the UTMB Organic Chemistry Core Laboratory, and its identity was verified by mass spectroscopy. Ni-NTA-purified TPR-MET (or cytoMET) was pre-incubated for 10 min at 4 °C in kinase reaction buffer (50 mM HEPES, pH 7.3, 100 mM NaCl, 25 mM MgCl2, 5 mM MnCl2, 5 mM ␤-glycerophosphate) with 1 ␮M ATP and 10 mM freshly prepared DTT added. Steady state rates were compiled to generate rate curves, and unweighted non-linear least squares regression analysis was used to determine Kmapp Determining this value at multiple inhibitor concentrations allowed calculation of KI by comparing to the previously calculated Km for ATP using the formula: KI ϭ [I]/((Kmapp/Km) Ϫ 1) [22]

RESULTS

DISCUSSION

TABLE III Inhibition kinetics of TPRMET and CytoMET

CytoMET p Value