Mitogenic signaling from the EGF receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism.

We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 mu M) augmented the EGF-induced [H-3]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 mu M) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine(654) was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.