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Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical

James R Broughman1, Limin Sun, Shahid Umar

  • 1Department of Integrative Biology, University of Texas Health Science Center, Houston, TX 77030, USA.

American Journal of Physiology. Gastrointestinal and Liver Physiology
|April 1, 2006
PubMed

Insights

Protein kinase C (PKC) activators alter cystic fibrosis transmembrane conductance regulator (CFTR) protein localization in intestinal cells. This suggests PKC-beta2 regulates CFTR

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Physiology

Background:

  • Cystic fibrosis transmembrane conductance regulator (CFTR) is crucial for ion transport.
  • Protein kinase C (PKC) is implicated in regulating CFTR function.
  • Understanding CFTR localization is key to understanding its regulation.

Purpose of the Study:

  • To investigate the effects of PKC-stimulating phorbol esters on CFTR expression and localization in HT-29 Cl.19A cell monolayers.
  • To determine the role of specific PKC isoforms, particularly PKC-beta2, in CFTR trafficking.

Main Methods:

  • Utilized immunofluorescence microscopy to assess CFTR and ZO-1 localization.
  • Quantified CFTR signal and puncta formation.
  • Measured CFTR mRNA levels and protein glycosylation.
  • Correlated CFTR changes with PKC activity and short-circuit current (ISC) measurements.

Main Results:

  • Phorbol esters (DOPPA, PMA) decreased luminal CFTR and increased cytoplasmic punctate structures.
  • DOPPA promoted CFTR puncta near tight junctions and ZO-1 accumulation.
  • PMA increased CFTR puncta size and PMA-induced CFTR loss correlated with PKC substrate phosphorylation.
  • Both agents reduced CFTR mRNA by 70% but minimally affected CFTR biosynthesis and glycosylation.
  • PKC-beta2 accumulation correlated with PMA- and DOPPA-induced inhibition of ISC.

Conclusions:

  • PKC activation alters CFTR subcellular localization, leading to reduced apical expression.
  • PKC-beta2 is hypothesized to play a significant role in regulating CFTR apical plasma membrane targeting.
  • These findings provide insights into the molecular mechanisms of CFTR regulation by PKC.

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