Mutations in the met Oncogene Unveil a “Dual Switch” Mechanism Controlling Tyrosine Kinase Activity

Abstract

The met oncogene, encoding the high affinity hepatocyte growth factor receptor, is the only known gene inherited in human cancer that is invariably associated with somatic duplication of the mutant locus. Intriguingly, mutated Met requires ligand stimulation in order to unleash its transforming potential. Furthermore, individuals bearing a germ line met mutation develop cancer only late in life and with incomplete penetrance. To date, there is no molecular explanation for this unique behavior, which is unusual for a dominant oncogene. Here we investigate the molecular mechanisms underlying met oncogenic conversion by generating antibodies specific for the differently phosphorylated forms of the Met protein. Using these antibodies, we show that activation of wild-type Met is achieved through sequential phosphorylation of Tyr 1235 and Tyr 1234 in the activation loop and that mutagenesis of either tyrosine dramatically impairs kinase function. Surprisingly, oncogenic Met mutants never become phosphorylated on Tyr 1234 despite their high enzymatic activity, and mutagenesis of Tyr 1234 does not affect their biochemical or biological function. By analyzing the enzymatic properties of the mutant proteins in different conditions, we demonstrate that oncogenic mutations do not elicit constitutive kinase activation but simply overcome the requirement for the second phosphorylation step, thus reducing the threshold for activation. In the presence of activating signals, these mutations result therefore in a dynamic imbalance toward the active conformation of the kinase. This explains why mutant met provides an oncogenic predisposition but needs a second activating "hit," provided by sustained ligand stimulation or receptor overexpression, to achieve a fully transformed phenotype.

Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase

“…However, phosphorylation at Tyr 1235 should require some motion of the activation loop to allow this residue enough freedom to serve as a substrate in a transphorylation reaction. Such a model is consistent with the observation that Tyr 1235 becomes phosphorylated ahead of Tyr 1234 during the activation process (39). However, phosphorylation at Tyr 1235 should only partially destabilize the autoinhibited conformation, because Tyr 1234 could still remain buried; this is consistent with the finding that phosphorylation at Tyr 1234 is required in addition to that at Tyr 1235 for full activation to occur (39).…”

“…Mutation of Tyr 1230 to His or Cys, found sporadically in cancers, should destabilize the inhibitory interactions made by the activation loop with the P-loop. Mutations of Asp 1228 to either Asn or His, found in hereditary renal cancer, should disrupt the salt bridge with Lys 1240, and therefore partially destabilize the autoinhibited conformation; this is consistent with the finding that such mutations, when present in the germ line, predispose the individual to cancer that still requires decades to appear (44), an increase in c-Met expression (26), and stimulation by HGF (28,39).…”

“…The juxtamembrane residues linked to the N terminus of the kinase participate in modulation of the signaling cascade through the recruitment of phosphatases (37) and ubiquitination complexes (38). Within the kinase domain itself, activation of the wild-type c-Met involves the required phosphorylation of two tyrosines in the activation loop, occurring stepwise, first at Tyr 1235 and following at Tyr 1234 (39). For activation of c-Met harboring oncogenic point mutations, the requirement for phosphorylation at Tyr 1234 can become lost (40,41).…”

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

“…MET receptor activity is regulated by phosphorylation of a number of sites including Y1234 and Y1235 in the activation loop, which are crucial for the regulation of kinase activity; the carboxy-terminal Y1349 and Y1356 in the multifunctional docking site required to recruit cytoplasmic signal transducers and adaptors, and S975 and Y1003 in the juxta-membrane region, phosphorylation of which causes MET ubiquitylation and degradation (5,8,14,15). On the basis of this understanding of MET signaling, we designed MET immunoassays to measure fulllength, transmembrane MET protein and three key phosphospecies involved in its signal transduction: pY1234/1235-MET, pY1235-MET, and pY1356-MET.…”

Functional analysis of HGF/MET signaling and aberrant HGF-activator expression in diffuse large B-cell lymphoma

“…In hereditary and sporadic papillary renal-cell cancer, most missense mutations are located in the tyrosine kinase domain of MET, causing constitutive activity and/or a lower threshold for HGFinduced activation of the tyrosine kinase. 39,41,56,57 Thus, the R1166Q mutant may have a similar effect. Since the JM region of MET harbors important negative regulatory sites involved in receptor ubiquitination, degradation, and inhibition of kinase activity, [58][59][60][61] the R988C MET mutation may affect these processes, leading to aberrant MET signaling.…”