Journal
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
Volume 307, Issue 10, Pages G992-G1001Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpgi.00180.2014
Keywords
apical recycling endosome; atypical protein kinase C; endothelial polarity; phosphoinositide-dependent protein kinase 1; small Ras-like guanosine 3 ',5 '-monophosphate
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Funding
- National Institute of Diabetes and Digestive and Kidney Diseases [R01-DK-087359, R01-DK-076652]
- [F32-DK-095503]
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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-2(BBe) 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-2(BBe) 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.
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