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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
08:07

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by

K P Windebank1, R T Abraham, G Powis

  • 1Department of Immunology, Mayo Clinic, Rochester, MN 55905.

Journal of Immunology (Baltimore, Md. : 1950)
|December 1, 1988
PubMed

Insights

Natural killer (NK) cells use inositol phosphates and calcium signaling for tumor cell killing. Cyclic AMP (cAMP) inhibits this process by disrupting NK cell receptor signaling.

Area of Science:

  • Immunology
  • Cellular Signaling

Background:

  • Natural killer (NK) cells are crucial for innate immunity, mediating direct cytotoxicity against tumor cells and antibody-coated targets via Fc receptor (FcR)-dependent mechanisms.
  • NK cell activation involves complex signaling pathways, including second messengers like inositol phosphates and intracellular calcium ions.

Purpose of the Study:

  • To investigate the role of phosphoinositide turnover and intracellular calcium in human NK cell cytotoxicity.
  • To determine how cyclic AMP (cAMP) signaling affects NK cell activation and phosphoinositide hydrolysis.

Main Methods:

  • Cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive and resistant tumor cells.
  • Measurements of inositol phosphates and intracellular free calcium concentration ([Ca2+]i) were performed.
  • The effect of Fc receptor (FcR) ligation with mAb 3G8 on phosphoinositide turnover was assessed.
  • NK cells were treated with forskolin to elevate intracellular cAMP levels and their impact on phosphoinositide hydrolysis was evaluated.

Main Results:

  • NK-sensitive target cells induced a significant rise in inositol phosphates and intracellular calcium in NK cells.
  • NK-resistant tumor cells failed to stimulate phosphoinositide production or elevate intracellular calcium.
  • FcR ligation also triggered phosphoinositide turnover, leading to rapid generation of inositol trisphosphate.
  • Elevated intracellular cAMP levels inhibited phosphoinositide hydrolysis induced by both target cells and FcR ligation in a dose-dependent manner.

Conclusions:

  • Phosphoinositide turnover is a critical early event in human NK cell-mediated cytotoxicity.
  • The inhibitory effect of cAMP on NK cell cytotoxicity may stem from its ability to uncouple NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.

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