title: GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells.
abstract: 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.
authors.affiliation: Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Andrea S
authors.initials: AS
authors.lastname: Oriolo
authors.forename: Flavia A
authors.initials: FA
authors.lastname: Wald
authors.forename: Gisella
authors.initials: G
authors.lastname: Canessa
authors.forename: Pedro J I
authors.initials: PJ
authors.lastname: Salas
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D019556:COS Cells; D016764:Cell Polarity; D002522:Chlorocebus aethiops; D015536:Down-Regulation / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006657:Histones / Q000235:genetics; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D014158:Transcription, Genetic; D014404:Tubulin / Q000378:metabolism
chemical_list: D006657:Histones; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C444456:TUBGCP6 protein, human; D014404:Tubulin; D007633:Keratins; D016203:CDC2 Protein Kinase