RESUMO
In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the gamma-tubulin ring complex, gamma-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in gamma-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced gamma-tubulin from the cytoskeleton and resulted in depolarization of gamma-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.
Assuntos
Células Epiteliais/metabolismo , Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Células COS , Polaridade Celular , Chlorocebus aethiops , Regulação para Baixo/genética , Células Epiteliais/citologia , Histonas/genética , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Centro Organizador dos Microtúbulos/metabolismo , Mutação/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Tubulina (Proteína)/metabolismoRESUMO
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.
Assuntos
Células Epiteliais/metabolismo , Filamentos Intermediários/metabolismo , Mucosa Intestinal/metabolismo , Fosfoproteínas/metabolismo , Animais , Células CACO-2 , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas do Citoesqueleto , Enterócitos/citologia , Enterócitos/metabolismo , Humanos , Mucosa Intestinal/citologia , Queratina-8 , Queratinas/biossíntese , Queratinas/genética , Camundongos , Camundongos Transgênicos , OctoxinolRESUMO
Atypical PKC (PKC iota) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKC iota 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 PKC iota is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKC iota 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 PKC iota.
Assuntos
Enterócitos/enzimologia , Proteínas de Choque Térmico HSP70/metabolismo , Filamentos Intermediários/enzimologia , Isoenzimas/metabolismo , Queratinas/metabolismo , Proteína Quinase C/metabolismo , Animais , Células CACO-2 , Proteínas de Choque Térmico HSP70/genética , Humanos , Isoenzimas/genética , Queratina-18/metabolismo , Queratina-19/metabolismo , Queratina-8/metabolismo , Queratinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Proteína Quinase C/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNARESUMO
Atypical protein kinase iota (PKCiota) 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. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota 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 PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.
Assuntos
Membrana Celular/enzimologia , Proteínas do Citoesqueleto/metabolismo , Mucosa Intestinal/enzimologia , Isoenzimas/metabolismo , Microdomínios da Membrana/enzimologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Células CACO-2 , Diferenciação Celular/fisiologia , Membrana Celular/ultraestrutura , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/química , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Humanos , Insetos , Mucosa Intestinal/citologia , Isoenzimas/genética , Microdomínios da Membrana/ultraestrutura , Camundongos , Microvilosidades/enzimologia , Microvilosidades/ultraestrutura , Fosforilação , Proteína Quinase C/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , Tirosina/metabolismoRESUMO
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 Caenorhabditis 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 keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Animais , Células Epiteliais/ultraestrutura , Exocitose/fisiologia , Humanos , Proteínas de Filamentos Intermediários/ultraestrutura , Filamentos Intermediários/ultraestrutura , Invertebrados/metabolismo , Queratinas/metabolismo , Queratinas/ultraestrutura , Mamíferos/metabolismo , Camundongos , Microtúbulos/metabolismo , Microtúbulos/ultraestruturaRESUMO
We have previously shown that microtubule-organizing centers (MTOCs) attach to the apical network of intermediate filaments (IFs) in epithelial cells in culture and in epithelia in vivo. Because that attachment is important for the architecture of microtubules (MTs) in epithelia, we analyzed whether chemical anoxia in LLC-PK1 and CACO-2 cells or unilateral ischemia-reperfusion in rat kidney (performed under fluorane anesthesia) had an effect on the binding and distribution of MTOCs. In culture, we found that chemical anoxia induces MTOC detachment from IFs by morphological and biochemical criteria. In reperfused rat proximal tubules, noncentrosomal MTOCs were fully detached from the cytoskeleton and scattered throughout the cytoplasm at 3 days after reperfusion, when brush borders were mostly reassembled. At that time, MTs were also fully reassembled but, as expected, lacked their normal apicobasal orientation. Two apical membrane markers expressed in S2 and S3 segments were depolarized at the same stage. At 8 days after reperfusion, membrane polarity, MTOCs, and MTs were back to normal. Na+-K+-ATPase was also found redistributed, not to the apical domain but rather to an intracellular compartment, as described by others (Alejandro VS, Nelson W, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling JD Jr., and Myers BD. Kidney Int 48: 1308-1315, 1995). The prolonged depolarization of the apical membrane may have implications in the pathophysiology of acute renal failure.