Anastasia Mashukova

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.

Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.

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.

Multiple roles for keratin intermediate filaments in the regulation of epithelial barrier function and apico-basal polarity

abstract As multicellular organisms evolved a family of cytoskeletal proteins, the keratins (types I and II) expressed in epithelial cells diversified in more than 20 genes in vertebrates. There is no question that keratin filaments confer mechanical stiffness to cells. However, such a number of genes can hardly be explained by evolutionary advantages in mechanical features. The use of transgenic mouse models has revealed unexpected functional relationships between keratin intermediate filaments and intracellular signaling. Accordingly, loss of keratins or mutations in keratins that cause or predispose to human diseases, result in increased sensitivity to apoptosis, regulation of innate immunity, permeabilization of tight junctions, and mistargeting of apical proteins in different epithelia. Precise mechanistic explanations for these phenomena are still lacking. However, immobilization of membrane or cytoplasmic proteins, including chaperones, on intermediate filaments (“scaffolding”) appear as common molecular mechanisms and may explain the need for so many different keratin genes in vertebrates.

The BAG-1 isoform BAG-1M regulates keratin-associated Hsp70 chaperoning of aPKC in intestinal cells during activation of inflammatory signaling

ABSTRACT Atypical PKC (&igr;/&lgr; and &zgr;; hereafter referred to as aPKC) is a key player in the acquisition of epithelial polarity and participates in other signaling cascades including the control of NF-&kgr;B signaling. This kinase is post-translationally regulated through Hsp70-mediated refolding. Previous work has shown that such a chaperoning activity is specifically localized to keratin intermediate filaments. Our work was performed with the goal of identifying the molecule(s) that block Hsp70 activity on keratin filaments during inflammation. A transcriptional screen allowed us to focus on BAG-1, a multi-functional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. We found the BAG-1 isoform BAG-1M upregulated threefold in human Caco-2 cells following stimulation with tumor necrosis factor receptor &agr; (TNF&agr;) to induce a pro-inflammatory response, and up to sixfold in mouse enterocytes following treatment with dextran sodium sulfate (DSS) to induce colitis. BAG-1M, but no other isoform, was found to co-purify with intermediate filaments and block Hsp70 activity in the keratin fraction but not in the soluble fraction within the range of concentrations found in epithelial cells cultured under control and inflammation conditions. Constitutive expression of BAG-1M decreased levels of phosphorylated aPKC. By contrast, knockdown of BAG-1, blocked the TNF&agr;-induced decrease of phosphorylated aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-&kgr;B.

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.

Conditional knockout of polarity complex (atypical) PKCι reveals an anti-inflammatory function mediated by NF-κB.

Atypical PKC, Par6, and Par3 constitute a conserved complex signaling cell asymmetry. In contrast to its role in other tissues, atypical PKC inhibits NF-κB activation in epithelia and may function in maintaining low levels of inflammation in addition to establishing apicobasal polarity.