Flavia A. Wald

Atypical protein kinase C (iota) activates ezrin 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 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.

Tumor Necrosis Factor Alpha and Inflammation Disrupt the Polarity Complex in Intestinal Epithelial Cells by a Posttranslational Mechanism

ABSTRACT 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-α) initiates an NF-κB-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-α 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-κB inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-α 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-α signaling.

Rescue of atypical protein kinase C in epithelia by the cytoskeleton and Hsp70 family chaperones.

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.

PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia

The polarity complex atypical PKC (aPKC) is rescued from degradation on intermediate filaments by Hsp70 chaperoning. The results indicate that PDK1 participates in the rescue mechanism and is localized to apical endosomes. Inhibition of dynamin-dependent endocytosis greatly decreases the steady-state levels of aPKC and Akt in their active conformation.

Aberrant expression of the polarity complex atypical PKC and non-muscle myosin IIA in active and inactive inflammatory bowel disease

Epithelial barrier function is contingent on appropriate polarization of key protein components. Work in intestinal epithelial cell cultures and animal models of bowel inflammation suggested that atypical PKC (aPKC), the kinase component of the Par3–Par6 polarity complex, is downregulated by pro-inflammatory signaling. Data from other laboratories showed the participation of myosin light chain kinase in intestinal inflammation, but there is paucity of evidence for assembly of its major target, non-muscle myosin II, in inflammatory bowel disease (IBD). In addition, we showed before that non-muscle myosin IIA (nmMyoIIA) is upregulated in intestinal inflammation in mice and TNFα-treated Caco-2 cells. Thus far, it is unknown if a similar phenomena occur in patients with IBD. Moreover, it is unclear whether aPKC downregulation is directly correlated with local mucosal inflammation or occurs in uninvolved areas. Frozen sections from colonoscopy material were stained for immunofluorescence with extensively validated specific antibodies against phosphorylated aPKC turn motif (active form) and nmMyoIIA. Inflammation was scored for the local area from where the material was obtained. We found a significant negative correlation between the expression of active aPKC and local inflammation, and a significant increase in the apical expression of nmMyoIIA in surface colon epithelia in inflamed areas, but not in non-inflamed mucosa even in the same patients. Changes in aPKC and nmMyoIIA expression are likely to participate in the pathogenesis of epithelial barrier function in response to local pro-inflammatory signals. These results provide a rationale for pursuing mechanistic studies on the regulation of these proteins.

Par-complex aPKC and Par3 cross-talk with innate immunity NF-κB pathway in epithelial cells

Summary Components of the Par-complex, atypical PKC and Par3, have been found to be downregulated upon activation of NF-&kgr;B in intestinal epithelial cells. To determine their possible role in pro-inflammatory responses we transduced Caco-2 human colon carcinoma cells with constitutively active (ca) PKC&igr; or anti-Par3 shRNA-expressing lentiviral particles. Contrary to previous reports in other cell types, ca-PKC&igr; did not activate, but rather decreased, baseline NF-&kgr;B activity in a luminiscence reporter assay. An identical observation applied to a PB1 domain deletion PKC&igr;, which fails to localize to the tight-junction. Conversely, as expected, the same ca-PKC&igr; activated NF-&kgr;B in non-polarized HEK293 cells. Likewise, knockdown of Par3 increased NF-&kgr;B activity and, surprisingly, greatly enhanced its response to TNF&agr;, as shown by transcription of IL-8, GRO-1, GRO-2 and GRO-3. We conclude that aPKC and Par3 are inhibitors of the canonical NF-&kgr;B activation pathway, although perhaps acting through independent pathways, and may be involved in pro-inflammatory responses.

p34cdc2-mediated phosphorylation mobilizes microtubule-organizing centers from the apical intermediate filament scaffold in CACO-2 epithelial cells.

We have shown previously that centrosomes and other microtubule-organizing centers (MTOCs) attach to the apical intermediate filament (IF) network in CACO-2 cells. In this cell line, intermediate filaments do not disorganize during mitosis. Therefore, we speculated that the trigger of the G2-M boundary may also detach MTOCs from their IF anchor. If that was the case, at least one of the proteins involved in the attachment must be phosphorylated by p34 cdc2 (cdk1). Using confocal microscopy and standard biochemical analysis, we found that p34 cdc2 -mediated phosphorylation indeed released MTOCs from IFs in permeabilized cells. In isolated, immunoprecipitated multiprotein complexes containing both γ-tubulin and cytokeratin 19, p34 cdc2 phosphorylated only one protein, and phosphorylation released cytokeratin 19 from the complexes. We conclude that this as yet unidentified protein is a part of the molecular mechanism that attaches MTOCs to IFs in interphase.

Ezrin phosphorylation in T567 is mediated by atypical PKC in intestinal epithelial cells: P-101.

Recent investigations have linked polymorphisms in the NOD2 gene with Crohn’s disease. This has sparked interest in developing experimental models appropriate for studying the role of NOD2 in the pathogenesis of Crohn’s disease. We assessed the response of various human intestinal epithelial cell lines to muramyl dipeptide (MDP), the ligand for NOD2. HCT116 cells, but not CaCo2 or HT29 cells, responded to MDP stimulation in a dose-dependent manner as assessed by the production of IL-8. This was consistent with the fact that transcript for NOD2 could be detected endogenously in HCT116 cells, but not CaCo2 or HT29 cells. No IL-8 was produced by HCT116 cells in response to the inactive D-D isomer of MDP. HCT116 cells also produced IL-8 in response to much lower doses of L18-MDP, a synthetic derivative of MDP with higher potency. The addition of MDP to HCT116 cells led to phosphorylation and degradation of IkBa, although over a prolonged period of time in comparison to the phosphorylation and degradation of IkBa in response to TNF. This was likely the result of increased IKK activity in HCT116 cells after MDP stimulation, which was detectable by kinase assay over a similar time course. The human colonic epithelial cell line HCT116 expresses NOD2 and can be activated by its ligand, MDP. Further, HCT116 cells have been reported to have high rates of homologous recombination. Together these characteristics make the HCT116 cell line an attractive choice for a model to investigate the role of NOD2 polymorphisms in Crohn’s disease using gene targeting.