Abstract. We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C3, (PLC3,) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement.Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, U73122 (1 #M) diminished both the EGF-induced motility and PLC responses, while its inactive analogue U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC3~-I fragment in EGFresponsive infectant lines. Lastly, anti-sense oligonucleotides (20 #M) to PLC3,-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC~/as the immediate post receptor effector in this motogenic pathway.We have demonstrated previously that EGFRmediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC3,-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate postreceptor stage, and we suggest that this is accomplished by differential activation of effector molecules. C ELL movement is essential for numerous normal biological and physiological events such as wound healing, fetal development, bone remodeling, angiogenesis, and the inflammatory response. Aberrant cell movement, on the other hand, contributes to the pathogenesis of many diseases, such as atherosclerosis and tumor invasion and metastasis. Cell movement is modulated by signals from extracellular environment. However, the intracellular signal Address all correspondence to Alan Wells, LHRB 531, Dept. of Pathology, University of Alabama at Birmingham, Birmingham, 975-9927. tra...
