Intensification of growth factor receptor signalling by phorbol treatment of ligand-primed cells implies a dimer-stabilizing effect of protein kinase C-dependent juxtamembrane domain phosphorylation.

Protein kinase C (PKC) phosphorylates the juxtamembrane domain of many growth factor receptors, but the physiologic effect of this modification on ligand signalling and desensitisation is unclear. Here we show that PKC-dependent transmodulation of EGFR and ErbB2 signalling is schedule-specific: prolonged pre-treatment of A431 cells with the PKC agonist phorbol dibutyrate potently inhibits subsequent ligand-induced EGFR signalling as expected, but EGF pre-treatment reverses the inhibitory effect of phorbol. The agonist activity of PKC on receptor signalling is even more apparent when cells are treated with phorbol in the presence of a tyrosine phosphatase inhibitor. Because these findings suggested a synergistic interaction between tyrosine- and PKC-dependent phosphorylation events, we sought to define the interactions of tyrosine-phosphorylated and PKC-modified ErbB2 subsets within EGF-inducible hetero-oligomers. Growth factor-dependent PKC transphosphorylation takes place exclusively within endocytosed tyrosine-phosphorylated receptor oligomers. Moreover, phorbol differentially affects two ErbB2 C-terminal autophosphorylation sites: whereas phosphorylation of Tyr1222 is reduced, phosphorylation of Tyr1139 is increased. These results suggest that PKC-dependent phosphorylation of the juxtamembrane domain may contribute positively to both internalisation and signalling of ligand-activated receptors, simultaneously accelerating termination of growth factor action. We propose that transient PKC-dependent signal amplification results from enhanced stability of liganded receptor oligomers due to phosphorylation-dependent juxtamembrane domain interactions, analogous to the protein-protein binding now known to be induced by serine-threonine phosphorylation of CREB and SMAD.