title: Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment.
abstract: Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.
authors.affiliation: Department of Pediatrics, Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut;
authors.forename: Dmitri
authors.initials: D
authors.lastname: Kravtsov
authors.affiliation: Department of Physiology, Nova Southeastern University, Ft. Lauderdale, Florida; Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida; and.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Mashukova
authors.affiliation: Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida; and.
authors.forename: Radia
authors.initials: R
authors.lastname: Forteza
authors.affiliation: Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida.
authors.forename: Maria M
authors.initials: MM
authors.lastname: Rodriguez
authors.affiliation: Department of Pediatrics, Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut;
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Ameen
authors.affiliation: Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida; and psalas@miami.edu.
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Salas
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018938:Caco-2 Cells; D016022:Case-Control Studies; D016764:Cell Polarity; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008286:Malabsorption Syndromes / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D008871:Microvilli / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D058990:Molecular Targeted Therapy; D009081:Mucolipidoses / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D024701:Myosin Type V / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014867:Water / Q000378:metabolism
chemical_list: C505832:MYO5B protein, human; D047428:Protein Kinase Inhibitors; D014867:Water; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D051057:Proto-Oncogene Proteins c-akt; C115015:PKC-3 protein; D011493:Protein Kinase C; D024701:Myosin Type V; D018995:Myosin Heavy Chains
keywords: apical recycling endosome; atypical protein kinase C; endothelial polarity; phosphoinositide-dependent protein kinase 1; small Ras-like guanosine 3′,5′-monophosphate