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Pull-down of Calmodulin-binding Proteins
07:51

Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

Oxidation of calmodulin alters activation and regulation of CaMKII

A J Robison1, Danny G Winder, Roger J Colbran

  • 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232-0615, USA.

Biochemical and Biophysical Research Communications
|March 9, 2007
PubMed

Insights

Oxidative stress impairs calmodulin function, affecting calcium signaling crucial for brain health. This damage hinders calmodulin's ability to regulate key proteins like CaMKII, potentially impacting neuronal and cardiac function.

Area of Science:

  • Biochemistry
  • Neuroscience
  • Cellular Biology

Background:

  • Reactive oxygen species and calcium dysregulation are linked to aging, ischemia, substance abuse, and neurodegenerative diseases.
  • Aged animals exhibit reduced oxidative repair, leading to increased methionine oxidation in calmodulin, a key calcium-signaling protein in the brain.

Purpose of the Study:

  • To investigate the functional consequences of calmodulin oxidation on its interaction with calcium/calmodulin-dependent protein kinase II (CaMKII).
  • To determine how oxidized calmodulin affects CaMKII activation, autophosphorylation, and binding to other proteins, and its implications for neuronal and cardiac function.

Main Methods:

  • In vitro biochemical assays to assess calmodulin's binding and activation of CaMKII.
  • Analysis of CaMKII interactions with NR2B subunits and alpha-actinin in the presence of oxidized calmodulin.
  • Comparison of oxidized calmodulin's effects on CaMKII versus its effects on the plasma membrane calcium ATPase (PMCA).

Main Results:

  • Oxidized calmodulin demonstrated an inability to activate CaMKII, support its autophosphorylation at specific sites (Thr286), or prevent autophosphorylation at other sites (Thr305/6).
  • Oxidized calmodulin failed to support CaMKII binding to the NR2B subunit of the NMDA receptor and could not effectively compete with alpha-actinin for CaMKII binding.
  • Unlike its interaction with PMCA, oxidized calmodulin showed reduced binding efficiency with CaMKII in rat brain lysates and in vitro.

Conclusions:

  • Oxidative stress significantly impairs calmodulin's ability to bind and regulate CaMKII, contrasting with its effects on PMCA.
  • The reduced functional capacity of oxidized calmodulin in regulating CaMKII interactions suggests a novel mechanism by which oxidative stress may disrupt neuronal and cardiac function.

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