Atypical PKC (PKCι) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKCι and the chaperone Hsp70.1 attached to the apical cytoskeleton. Pull-down experiments using purified and recombinant proteins showed a complex of Hsp70 and atypical PKC on filamentous keratins. Transgenic animals overexpressing keratin 8 displayed delocalization of Hsp70 and atypical PKC. Two different keratin-null mouse models, as well as keratin-8 knockdown cells in tissue culture, also showed redistribution of Hsp70 and a sharp decrease in the active form of atypical PKC, which was also reduced by Hsp70 knockdown. An in-vitro turn motif rephosphorylation assay indicated that PKCι is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKCι only when supplemented with the cytoskeletal pellet or filamentous highly purified keratins, a function abolished by immunodepletion of Hsp70 but rescued by recombinant Hsp70. We conclude that both filamentous keratins and Hsp70 are required for the rescue rephosphorylation of mature atypical PKC, regulating the subcellular distribution and steady-state levels of active PKCι.

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 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. PKCι phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human 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 the PKA regulatory subunit. Expression of a dominant-negative form of PKCι also decreased T567-P signal, and expression of a constitutively active PKCι mutant showed depolarized distribution of T567-P. 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.

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models, have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and C. elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratinnull or overexpression phenotypes are still missing, it is hoped investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.

Inflammatory processes disrupt the barrier function in epithelia. Increased permeability often leads to chronic of inflammation. Important among other cytokines, tumor necrosis factor alpha (TNF-␣) initiates an NF-B-mediated response that leads to upregulation of myosin light chain kinase (MLCK), a hallmark of the pathogenesis of inflammatory bowel disease. Here, we found that two components of the evolutionarily conserved organizer of tight junctions and polarity, the polarity complex (atypical protein kinase C [aPKC]-PAR6-PAR3) were downregulated by TNF-␣ signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Decreases in aPKC levels were due to decreased chaperoning activity of Hsp70 proteins, with failure of the aPKC rescue machinery, and these effects were rescued by NF-B inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-␣ signaling, including apical nonmuscle myosin II accumulation and myosin light chain phosphorylation. These effects, including ZO-1 downregulation, were rescued by overexpression of constitutively active aPKC. We conclude that this novel mechanism is a complementary effector pathway for TNF-␣ signaling.

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