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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

Effect of inositol hexakisphosphate kinase 2 on transforming growth factor beta-activated kinase 1 and NF-kappaB

Bei H Morrison1, Joseph A Bauer, Joseph A Lupica

  • 1Center for Hematology and Oncology Molecular Therapeutics, Taussig Cancer Center, and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.

The Journal of Biological Chemistry
|March 24, 2007
PubMed

Insights

Inositol hexakisphosphate kinase 2 (IHPK2) suppresses tumor growth by binding TRAF2, inhibiting NF-kappaB signaling, and promoting apoptosis, partly independent of its kinase activity.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Oncology

Background:

  • Inositol hexakisphosphate kinase 2 (IHPK2) is a kinase that promotes apoptosis and suppresses growth during cellular stress.
  • Previous studies showed IHPK2 overexpression sensitizes ovarian carcinoma cells to interferons and gamma-irradiation.
  • A kinase-dead IHPK2 mutant retained partial apoptosis-inducing function, suggesting a kinase-independent mechanism.

Purpose of the Study:

  • To investigate the kinase-independent, death-promoting functions of IHPK2.
  • To elucidate the interaction between IHPK2 and tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2).
  • To determine the role of IHPK2-TRAF2 binding in regulating NF-kappaB signaling and apoptosis.

Main Methods:

  • Transfection of ovarian carcinoma cells with wild-type IHPK2 and specific mutants (S347A, S359A).
  • Assessment of IHPK2 binding to TRAF2.
  • Measurement of transforming growth factor beta-activated kinase 1 (TAK1) phosphorylation and NF-kappaB DNA binding activity following TNF-alpha stimulation.
  • Evaluation of apoptosis using terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay.

Main Results:

  • IHPK2 binds to TRAF2, with Ser-347 and Ser-359 identified as crucial binding sites.
  • Mutations at Ser-347 and Ser-359 (S347A/S359A) abrogated IHPK2's ability to inhibit TAK1 phosphorylation and NF-kappaB signaling.
  • Cells expressing wild-type IHPK2 or non-inhibitory mutants showed increased apoptosis upon TNF-alpha treatment compared to cells with the S347A/S359A mutant.
  • IHPK2-TRAF2 binding inhibits TAK1/NF-kappaB signaling, contributing to IHPK2-mediated apoptosis.

Conclusions:

  • IHPK2 interacts with TRAF2 through specific serine residues (Ser-347, Ser-359).
  • This interaction inhibits the TAK1/NF-kappaB pathway, a mechanism partially responsible for IHPK2's pro-apoptotic function.
  • IHPK2 possesses both kinase-dependent and kinase-independent functions in regulating cell death and growth suppression.

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