title: PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia.
abstract: Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.
authors.affiliation: Department of Cell Biology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Mashukova
authors.forename: Radia
authors.initials: R
authors.lastname: Forteza
authors.forename: Flavia A
authors.initials: FA
authors.lastname: Wald
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Salas
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D011992:Endosomes / Q000201:enzymology*; D020895:Enterocytes / Q000166:cytology / Q000201:enzymology*; D055785:Gene Knockdown Techniques; D006801:Humans; D007382:Intermediate Filaments / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; C115015:PKC-3 protein; D011493:Protein Kinase C