Localized stimulation of the EGF receptor

Abstract

In cancer, EGF plays a substantial role in inducing tumor cell movement. However, the mechanisms by which cells respond to concentration gradients of this chemoattractant are poorly understood. To model this process, we have locally stimulated cells with EGF-coated beads. We have seen localized, phase dense protrusions where the EGF-bead contacts the cell. By determining the radius of immunostaining at the bead site, a spatial scale was elucidated where phosphorylated receptor was detected closest to the bead. N-WASP, p34 and cofilin, all critical components for inducing actin polymerization, were also associated at the bead site. F-actin and phospho-Akt staining were broader. Cortactin staining was not visible at the bead site, but it was present throughout protrusions. The only signal seen throughout the cell after EGF-bead addition was phospho-ERK. Therefore, actin polymerization is intimately associated with EGF-bead attachment to the cell and a long-range signal through ERK activation is also noticeable using this method of cell stimulation.;Using various inhibitors it was shown that this EGF-bead induced response was dependent on EGFR kinase activity, but not PI3-K or RhoGTPases. Knocking down either cofilin or p34 inhibited the EGF-bead response by close to fifty percent. When both proteins were simultaneously knocked down, the inhibition was almost complete, indicating that these two proteins work independently in parallel pathways in this actin polymerization response. To understand the Arp2/3-dependent pathway of EGF-induced actin polymerization, we inhibited the expression of Arp2/3-activating proteins. N-WASP appeared to be important, whereas WAVE loss did not have an effect. We then knocked down activators of N-WASP, yielding Grb2 and Nck2 as required in the EGF-bead response. Interestingly, inhibition of cortactin expression resulted in an enhancement of protrusion responses and cortactin overexpression inhibited the response. We found that cortactin can restrict N-WASP localization to the EGF-bead. In addition, cortactin-deficient cells have increased lamellipod dynamics, but show reduced net cell translocation. Rather than initiating actin polymerization, this data suggests that cortactin's major role is to control the extent of Arp2/3 activation by N-WASP and to incorporate these freshly made actin filaments into the cell's original cytoarchitecture resulting in consistent protrusive forces.