SIGNALING FROM TPA TO MAP KINASE REQUIRES PROTEIN-KINASE-C, RAF AND MEK - RECONSTITUTION OF THE SIGNALING PATHWAY IN-VITRO

The phorbol ester PMA/TPA (phorbol 12-myristate 13-acetate) is a poten t tumor promoter which mimicks distinct intracellular signalling event s triggered by activated growth factor receptors, e.g. the activation of MAP kinases. The largest known family of TPA-binding proteins compr ise members of the protein kinase C (PKC) family although other TPA-bi nding proteins outside the PKC family have recently been identified. I n this report we addressed the mechanism and the pathway by which TPA induces the activation Of MAPkinases. Using recombinant proteins and i n vitro phosphorylation reactions we identified the components in the signal transduction pathway from TPA to MAPkinase and we show that the activation of MAPkinase by TPA requires the presence of protein kinas e C, c-raf and the MAPkinase activator MEK. We also find that the acti vation of raf autophosphorylation in vitro correlates with the ability of Raf to signal to MAPkinase. Thus the activation of Raf by PKC appa rently can trigger the same signalling pathway as oncogenic Raf or Raf activation by vas in combination with tyrosine phosphorylation.