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First published online 12 February 2008
doi: 10.1242/jcs.016246

Journal of Cell Science 121, 644-654 (2008)
Published by The Company of Biologists 2008
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Research Article

Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells

Flavia A. Wald1, Andrea S. Oriolo1, Anastasia Mashukova1, Nevis L. Fregien1, Amber H. Langshaw2 and Pedro J. I. Salas1,*

1 Department of Cell Biology and Anatomy
2 Department of Pediatrics, University of Miami Miller School of Medicine, RMSB 4090–R124, 1600 NW 10th Avenue, Miami, FL 33135, USA

* Author for correspondence (e-mail: psalas{at}med.miami.edu)

Accepted 5 December 2007

Atypical protein kinase iota (PKC{iota}) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of 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 T567-P during enterocyte differentiation in vivo. PKC{iota} phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKC{iota} 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 PKC{iota}-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKC{iota} also decreased T567-P signal, and expression of a constitutively active PKC{iota} mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKC{iota} is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.

Key words: ERM proteins, Apical domain, Brush border, Epithelial polarity






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