p34 -mediated Phosphorylation Mobilizes Microtubule-organizing Centers from the Apical Intermediate Filament Scaffold in CACO-2 Epithelial Cells

Terán-Figueroa, Yolanda; Wald, Flavia A.; Salas, Pedro J.

doi:10.1074/jbc.m207037200

Cited by 5 publications

(13 citation statements)

References 27 publications

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“…To this end, we immunoprecipitated intermediate filaments under nondenaturing conditions in small fragments separated by sucrose gradients (Rodriguez et al, 1994;Salas, 1999;Figueroa et al, 2002). Although the yield is poor and the proportion of fragments useful for immunoprecipitation is relatively small (top half of the gradient), we demonstrated that these fragments represent a random sampling of the cytoskeletal preparation (Salas, 1999).…”

Section: Triton X-100-insoluble Ezrin Occurs In An Apical Submembranementioning

confidence: 99%

“…Keratin knockdowns in CACO-2 cells showed shorter and sparser microvilli (Salas et al, 1997), but the microvillus phenotype in K8-null mice was only marginal (Ameen et al, 2001). The molecular mechanism involved in the disruption of the orientation of microtubules has been pinpointed to a function of IFs attaching microtubule organizing centers (MTOCs) (Salas, 1999;Figueroa et al, 2002). The mechanism by which IF down-regulation affected the development of apical microvilli in CACO-2 cells, however, remained obscure.…”

Section: Introductionmentioning

confidence: 99%

“…Extraction in TX-100 (PBS supplemented with 1% TX-100, 2 mM EGTA, 1 mM MgCl 2 , 70 nM phalloidin, and a cocktail of antiproteases), sonication, isolation, and immunoprecipitation of the Triton-insoluble fragments of the cytoskeleton have been described previously (Salas, 1999). Double immunoprecipitation was described previously (Figueroa et al, 2002). Briefly, the antibodies were covalently attached to CNBr-activated Sepharose (Amersham Biosciences, Piscataway, NJ).…”

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confidence: 99%

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Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Wald

Oriolo

Casanova

et al. 2005

MBoC

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Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells. INTRODUCTIONEzrin, a member of the ezrin-radixin-moesin (ERM) family, is a conspicuous component of the apical F-actin-based scaffold in simple epithelial cells. It connects NHERF/EBP-50 (which in turn binds important membrane proteins such as CFTR and NHE-3) to F-actin and also enables protein kinase A-mediated regulation of apical channels (Kurashima et al., 1999;Louvet-Vallée, 2000;Weinman et al., 2000Weinman et al., , 2001. Because ezrin is the earliest protein of this complex scaffold to be recruited, it has long been considered as an organizer of the brush border (Bretscher et al., 1997). In support of that view, ezrin knockout mice show a distinctive brush-border phenotype with much shorter microvilli that resemble undifferentiated stages (Saotome et al., 2004). Furthermore, expression of ezrin in fibroblasts is sufficient to organize microvilli (Shaw et al., 1998). After translation, ezrin initially adopts a "dormant" configuration, in which the C-and N-terminal domains bind to each other. On phosphorylation in T567, it changes to an open "active" configuration, freeing the C-terminal domain (C-ERMAND) to bind F-actin and the N-terminal domain (N-ERMAND) to bind NHERF or transmembrane proteins (Bretscher et al., 2000). Although the mechanism of activation and assembly of ezrin has been studied in great detail by several groups, the mechanisms that ensure that ezrin assembly occur mostly under the apical membrane of simple epithelial cells remain obscure. Fievet et al. (2004) have shown that binding to phosphatidylinositol 4,5-diphosphate (PIP 2 ) is a prerequisite for activation. However, PIP 2 is not known to be apically polarized in epithelial cells, rather it is basol...

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Section: Triton X-100-insoluble Ezrin Occurs In An Apical Submembranementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Wald

Oriolo

Casanova

et al. 2005

MBoC

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“…Conversely, the molecular basis of the apical polarization of keratin IFs in simple epithelial cells is currently unknown. However, unlike in the case of keratinocyte IFs [37] or neurofilament IFs [38] that move on microtubules, the steady-state, polarized, distribution of keratin IF in simple epithelial cells is not affected by long and extensive depolymerization of MTs, such as in the case of ATP depletion in tissue culture cells, ischemia-reperfusion in kidney proximal tubules [17] or long incubations in anti-microtubular drugs [39]. Moreover, in simple epithelia IFs are not perinuclear, with the exception of a few lateral bundles coming in contact with the lateral aspect of the nucleus (Fig.…”

Section: Location Location Location: Apical Distribution Of Intermementioning

confidence: 99%

“…Furthermore, this colocalization can be disrupted by phosphorylation mediated by recombinant active Cdk1(p34 cdc2 )/cyclin B in interphasic cells, and is naturally lost in mitotic cells. Cdk1 phosphorylates a 190 kDa protein that co-immunoprecipitates with γ-tubulin [39]. In addition, the apical localization of centrosomes and non-centrosomal γ-TurCs is abrogated by keratin knock-down in tissue culture cells [43] as well as in villus enterocytes in K8-null mice [28].…”

Section: Role Of Keratin Ifs In the Subcellular Localization Of Micromentioning

confidence: 99%

Intermediate filaments: A role in epithelial polarity

Oriolo

Wald

Palau

et al. 2007

Experimental Cell Research

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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.

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Protein Sorting in the Exocytic and Endocytic Pathways in Polarized Epithelial Cells

Hunt

Nelson

2006

Physiology of the Gastrointestinal Tract

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p34 -mediated Phosphorylation Mobilizes Microtubule-organizing Centers from the Apical Intermediate Filament Scaffold in CACO-2 Epithelial Cells

Cited by 5 publications

References 27 publications

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Intermediate filaments: A role in epithelial polarity

Protein Sorting in the Exocytic and Endocytic Pathways in Polarized Epithelial Cells

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