Rescue of Atypical PKC: Role of Hsp70 Chaperone and Intermediate Filament Cytoskeleton

Objective. In this study we tested the hypothesis that a pool of atypical PKC is associated with Hsp70 and intermediate filament scaffold in polarized epithelial cells. Background. The Protein Kinase C (PKC) family in humans comprises 10 isoforms categorized in 3 subclasses: conventional, novel and atypical. Atypical PKC is a key regulator of cell assymetry organization. Unlike other kinases, PKC isozymes lose their activation phosphorylation in a substrate-dependent manner, i.e. they become inactivated as a consequence of their own function. Once dephosphorylated, the PKC molecules are ubiquitinylated and degraded. Hsp70 chaperone was shown to bind dephosphorylated conventional PKC isoforms and rescue them from degradation to regain function. A role of Hsp70 in rescuing atypical PKC (aPKC) has not been established. However, based on the presence of the invariant Leu motif necessary for Hsp70 binding, we suggested that functional pool of aPKC is also maintained by Hsp70. Methods. We performed confocal colocalization experiments, immunoblot analysis, qPCR and shRNA-mediated knockdown of Hsp70 and keratins in Caco-2 (human colon carcinoma) epithelial cells. Results. Our experiments demonstrate that Hsp70 and aPKC exist in two different pools: soluble and associated to the cytoskeleton. Both Hsp70 and the native filamentous keratin cytoskeleton are necessary for the rephosphorylation of the inactive form of aPKC and its rescue from degradation. Conclusion. Our results not only confirmed the association of aPKC with Hsp70, but also showed a novel function of the 16 cytoskeleton in the post-translational regulation of aPKC expression and localization.