Novel mechanisms of c-erbB-2 activation

C-erbB tyrosine kinase receptor signalling preceded by ligand-induced dimerization represents an important mechanism controlling cell proliferation in normal physiology and malignant diseases. The orphan c-erbB-2 receptor reveals high tyrosine kinase activity and is the major dimerization partner for the other c-erbB receptors. Data are presented showing that the kinetics of c-erbB-1 and c-erbB-2 receptor autophosphorylation have a transient and a stable component and are compared to the dynamics of heterodimer formation, which corresponded to the stable/slow component. These and previous data obtained in the host laboratory, demonstrating lateral signal propagation for c-erbB-1, cannot be fully explained by the classical homo- or heterodimerization models. The current proposal aims at elucidating the mechanism of c-erbB-2 signalling in the c-erbB-1/c-erbB-2 system. In order to achieve this aim, FRET experiments will compare the kinetics of dimerization and phosphorylation. Autophosphorylation of c-erbB-1 and c-erbB-2 will be examined in live cells using novel autophosphorylation sensors. Kinase-dead mutants of these sensors will be used to analyse the relative contribution of the c-erbB-1 and c-erbB-2 to the two components. Inhibition of tyrosine phosphatase activity by activated c-erbB-1 was previously shown to be responsible for lateral signal propagation on the c-erbB-1 receptor, probably due to the production of reactive oxygen species (ROS). Therefore inhibition of ROS production or scavenging of ROS will be performed to evaluate ROS as possible candidates for the observed early c-erbB-2 receptor phosphorylation in absence of heterodimerization.