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The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation
L Naldini1, E Vigna, R Ferracini
1Department of Biomedical Sciences and Human Oncology, University of Turin Medical School, Italy.
Abstract:
Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.
Insights
Tyrosine autophosphorylation powerfully activates the MET receptor tyrosine kinase. This activation increases enzyme efficiency (Vmax) without altering substrate binding (Km), highlighting a key regulatory mechanism in cell proliferation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Protein tyrosine kinases regulate cell proliferation and their activity is critical in normal and cancerous cells.
- The MET oncogene encodes a transmembrane receptor-like tyrosine kinase with a unique disulfide-linked heterodimeric structure.
Purpose of the Study:
- To investigate the mechanism by which the MET receptor tyrosine kinase activity is regulated.
- To determine the role of tyrosine autophosphorylation in the activation of MET kinase activity.
Main Methods:
- Quantification of MET kinase activity enhancement using phosphorylation assays with exogenous substrates.
- Kinetic analysis, including Vmax and Km determination, to characterize the effect of autophosphorylation.
- Investigation of ATP dependence, substrate specificity, and phosphatase treatment to establish causality.
Main Results:
- Tyrosine autophosphorylation was shown to powerfully activate the tyrosine kinase activity of the MET-encoded protein.
- Activation resulted in an increased Vmax for phosphotransfer, with no change in Km for the substrate.
- Autophosphorylation occurred on a specific tryptic peptide, likely via an intermolecular reaction, within the intracellular domain.
Conclusions:
- Tyrosine autophosphorylation is a crucial positive modulator of MET receptor tyrosine kinase activity.
- The activation mechanism involves enhanced catalytic efficiency, regulated by the intracellular moiety of the MET protein.
- Understanding this activation pathway is vital for comprehending MET-mediated cell proliferation and oncogenesis.