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Vol. 18, Issue 3, 781-794, March 2007

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GCP6 Binds to Intermediate Filaments: A Novel Function of Keratins in the Organization of Microtubules in Epithelial Cells

Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL 33136

Submitted March 14, 2006; Revised November 3, 2006; Accepted November 5, 2006
Monitoring Editor: M. Bishr Omary

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 -tubulin ring complex, -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 -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 -tubulin from the cytoskeleton and resulted in depolarization of -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.

Address correspondence to: Pedro J.I. Salas (psalas{at}miami.edu)

Abbreviations used: -TurC, -tubulin ring complex; Cdk, cyclin-dependent kinase; dox, doxycycline; HA, hemagglutinin; IF, intermediate filament; K, keratin; MT, microtubule; TET, tetracycline; TX-, Triton X-.

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