Atypical Protein Kinase C (iota) mediates local ezrin activation in the apical domain of intestinal epithelial cells

Atypical Protein Kinase iota (PKCι) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation in T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of pT567 during enterocyte differentiation in vivo . PKCι phosphorylated ezrin in T567 in vitro , and in Sf9 cells that do not activate h-ezrin. In CACO-2 human intestinal cells in culture, PKCι co-immunoprecipitated with ezrin and was knocked-down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCι-depleted cells showed fewer and shorter microvilli and redistribution of PKA regulatory subunit. Expression of a dominant negative form of PKCι also decreased pT567 signal, and expression of a constitutively active PKCι mutant showed depolarized distribution of pT567. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCι is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.