Mutations in the met Oncogene Unveil a "Dual Switch" Mechanism Controlling Tyrosine Kinase Activity *

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 Tyr1235 and Tyr1234 in the activation loop and that mutagenesis of either tyrosine dramatically impairs kinase function. Surprisingly, oncogenic Met mutants never become phosphorylated on Tyr1234 despite their high enzymatic activity, and mutagenesis of Tyr1234 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

Nanoroughness, Surface Chemistry and Drug Delivery Control by Atmospheric Plasma Jet on Implantable Devices

Implantable devices need specific tailored surface morphologies and chemistries to interact with the living systems or to actively induce a biological response also by the release of drugs or proteins. These customised requirements foster technologies that can be implemented in additive manufacturing systems. Here we present a novel approach based on spraying processes that allows to control separately topographic features in the submicron range ( 3d 60 nm - 2 \ub5m), ammine or carboxylic chemistry and fluorophore release even on temperature sensitive biodegradable polymers such as polycaprolactone (PCL). We developed a two-steps process with a first deposition of 220 nm silica and poly(lactic-co-glycolide) (PLGA) fluorescent nanoparticles by aerosol followed by the deposition of a fixing layer by atmospheric pressure plasma jet (APPJ). The nanoparticles can be used to create the nano-roughness and to include active molecule release, while the capping layer ensures stability and the chemical functionalities. The process is enabled by a novel APPJ which allows deposition rates of 10 - 20 nm\ub7s-1 at temperatures lower than 50 \ub0C using argon as process gas. This approach was assessed on titanium alloys for dental implants and on PCL films. The surfaces were characterized by FT-IR, AFM and SEM. Titanium alloys were tested with pre-osteoblasts murine cells line, while PCL film with fibroblasts. Cell behaviour was evaluated by viability and adhesion assays, protein adsorption, cell proliferation, focal adhesion formation and SEM. The release of a fluorophore molecule was assessed in the cell growing media, simulating a drug release. Osteoblast adhesion on the plasma treated materials increased by 20% with respect to commercial titanium alloys implants. Fibroblast adhesion increased by a 100% compared to smooth PCL substrate. The release of the fluorophore by the dissolution of the PLGA nanoparticles was verified and the integrity of the encapsulated drug model confirmed