Rescue of atypical protein kinase C in epithelia by the cytoskeleton and Hsp70 family chaperones

Abstract

Atypical PKC (PKCι) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKCι 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 PKCι is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKCι 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 PKCι.

Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C

“…2A, arrowheads, and data not shown), pointing to an unrecognized role for Hsp70/Hsc70 in promoting degradation of the activated protein. In addition, consistent with the known role of Hsp70/ Hsc70 activity in facilitating rephosphorylation of activated PKCs (58,59,67), PES induced accumulation of faster migrating dephosphorylated forms of PKC␣ ( Fig. 2A, arrow).…”

“…1E) and mutation of these sites leads to unstable/inactive enzyme (5-7). However, recent studies by several groups have determined that rephosphorylation of activated, dephosphorylated PKCs requires Hsp70 activ- ity (58,59,67). Thus, Hsp70/Hsc70 inhibition can be used to assess the extent to which rephosphorylation accounts for the failure to detect substantive amounts of nonphosphorylated PKC␣ in agonist-treated cells.…”

Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size – implication for FasR-associated apoptosis

“…Considering that different PKC isoforms can interact with K8/K18 IFs (Bordeleau et al, 2010;Mashukova et al, 2009;Omary et al, 1992), and that PKCδ modulates ASMase activation (Zeidan and Hannun, 2007), we first compared the surface membrane translocation of PKCα, PKCδ and PKCε, which is associated with their activations in the present K8-null hepatocyte model. At 52 h post seeding, WT and K8-null hepatocytes were transfected with a cDNA encoding EGFP-tagged PKCα, PKCδ and PKCε ( Fig.…”

“…plectin) and a scaffolding protein (e.g. receptor of activated C kinase 1, RACK1) in spreading or migrating keratinocytes and hepatocytes (Bordeleau et al, 2010;Mashukova et al, 2009;Osmanagic-Myers and Wiche, 2004). In this context, keratin IFs can be part of a cytoskeleton-dependent signaling platform for PKCδ in epithelial cells, in response to surface receptor stimulation.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…This interaction stabilises PKC and facilitates re-phosphorylation at this site and thus helps to preserve the function of these enzymes [67,68].…”