Myosin 5b loss of function leads to defects in polarized signaling: Implication for microvillus inclusion disease pathogenesis and treatment

Dmitri Kravtsov, Anastasia Mashukova, Radia Forteza, Maria M. Rodriguez, Nadia A. Ameen, Pedro J. Salas

Research output: Contribution to journalArticle

23 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)G992-G1001
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume307
Issue number10
DOIs
StatePublished - Nov 15 2014

Keywords

  • Apical recycling endosome
  • Atypical protein kinase C
  • Endothelial polarity
  • Phosphoinositide-dependent protein kinase 1
  • Small ras-like guanosine 3ʹ,5ʹ-monophosphate

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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