Rac regulates the interaction of fascin with protein kinase C in cell migration

Abstract

Fascin is an actin-bundling protein that is low or absent in normal epithelia; its upregulation correlates with poor prognosis in many human carcinomas. We have recently demonstrated in mouse xenograft models that fascin contributes to tumour development and metastasis through its dual actin-bundling and active PKC-binding activities. Rac was implicated as a regulator of fascin-dependent colon carcinoma cell migration in vitro. Here, we tested the hypothesis that Rac regulates the interaction of fascin with active PKC. The major conventional PKC in colon carcinoma cells is protein kinase Cγ (PKCγ). Endogenous PKCγ, fascin and Rac1 colocalised at lamellipodial margins of migrating cells. Colocalisation of fascin and PKCγ depended on Rac activity, and inhibition of Rac decreased PKCγ activity in cell extracts but not in vitro. Fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy uncovered that fascin and PKCγ interact in protrusions and filopodia of migrating cells. Mechanistically, the interaction depended on phosphorylated fascin, active PKCγ and active Rac, but not on active Cdc42. The activity of Rac on the fascin/ PKC complex was mediated in part by Pak. Elucidation of this novel pathway for regulation of the fascin/PKCγ complex in migrating carcinoma cells suggests novel targets for therapeutic intervention in metastasis.