GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells

• Published in: Molecular biology of the cell Vol. 18; no. 3; pp. 781 - 794
• Main Authors: Oriolo, Andrea S, Wald, Flavia A, Canessa, Gisella, Salas, Pedro J I
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.03.2007
• Subjects: Animals; CDC2 Protein Kinase - metabolism; Cell Polarity; Cercopithecus aethiops; COS Cells; Down-Regulation - genetics; Epithelial Cells - cytology; Epithelial Cells - metabolism; Histones - genetics; Humans; Intermediate Filaments - metabolism; Keratins - metabolism; Mice; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Microtubule-Organizing Center - metabolism; Microtubules - metabolism; Mutation - genetics; Phosphorylation; Promoter Regions, Genetic - genetics; Protein Binding; Protein Transport; RNA, Small Interfering - metabolism; Transcription, Genetic; Tubulin - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E06-03-0201

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.