THE RI-ALPHA SUBUNIT OF PROTEIN-KINASE-A (PKA) BINDS TO GRB2 AND ALLOWS PKA INTERACTION WITH THE ACTIVATED EGF-RECEPTOR

Functional interactions between protein kinase A (PKA) and epidermal g rowth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or i ts regulatory submit RI alpha are generally overexpressed in cancer ce lls and are induced following transforming growth factor alpha (TGF al pha)/EGF-R-dependent transformation. Downregulation of RI alpha/PKAI i nhibits TGF alpha expression and EGF-R-dependent signalling. We have p reviously shown that addition of EGF to quiescent human normal epithel ial MCF-10A cells determines PKAI expression and cell membrane translo cation before cells enter S phase, while PKAI inhibition prevents S ph ase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstr ate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A ce lls, Moreover, induction of mitogen-activated protein kinase (MAPK) ac tivity following EGF-R activation is mimicked by PKAI overexpression a nd inhibited by downregulators of PKAI. Finally, the PKAI-EGF-R associ ation occurs through the binding of RI alpha to the SH3 domain(s) of G rb2 adaptor protein, thus allowing the recruitment of the PKAI holoenz yme to the activated EGF-R. This is the first demonstration of a direc t interaction of PKAI with the activated EGF-R macromolecular signalli ng complex.