Oligomerization-dependent changes in the thermodynamic properties of the TPR-MET receptor tyrosine kinase.
Although oligomerization of receptor tyrosine kinases (RTKs) is necessary for receptor activation and signaling, a quantitative understanding of how oligomerization mediates these critical processes does not exist. We present a comparative thermodynamic analysis of functionally active dimeric and functionally inactive monomeric soluble analogues of the c-MET RTK, which clearly reveal that oligomerization regulates the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. Thermodynamic binding data for oligomeric c-MET were obtained from the dimeric TPR-MET oncoprotein, a functionally active fusion derivative of the c-MET RTK. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil dimerization domain from the nuclear pore complex. Comparative data were obtained from a soluble monomeric kinase compromising the c-MET cytoplasmic domain (cytoMET). Significantly, under equilibrium binding conditions, the oligomeric phosphorylated kinase showed a significantly lower dissociation constant (K(d,dimer) = 11 microM) for a tyrosine-containing peptide derived from the C-terminal tail of the c-MET RTK when compared to the phosphorylated monomeric kinase cytoMET (K(d,monomer) = 140 microM). Surprisingly, equilibrium dissociation constants measured for the kinase and ATP were independent of the oligomerization state of the kinase (approximately 10 microM). Stopped-flow analysis of peptide substrate binding showed that the association rate constants (k(2)) differed 2-fold and dissociation rate constants (k(-2)) differed 10-fold when phosphorylated TPR-MET was compared to phosphorylated cytoMET. ATP binding abrogated the differences in k(2) rates observed between the two oligomeric states of the c-MET cytoplasmic domain. These results clearly imply that oligomerization induces important thermodynamic and conformational changes in the substrate binding regions of the c-MET protein and provide quantitative mechanistic insights into the necessary role of oligomerization in RTK activation.
Department of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555-0645, USA.
John L
JL
Hays
Stanley J
SJ
Watowich
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology*; D019281:Dimerization; D006801:Humans; D007313:Insecta; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D019859:Proto-Oncogene Proteins c-met / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D019859:Proto-Oncogene Proteins c-met