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Pull-down of Calmodulin-binding Proteins
07:51

Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase,

N Sahyoun1, O B McDonald, F Farrell

  • 1Wellcome Research Laboratories, Research Triangle Park, NC 27709.

Proceedings of the National Academy of Sciences of the United States of America
|April 1, 1991
PubMed

Insights

CaM kinase Gr selectively phosphorylates Rap-1b, a brain protein, linking neuronal signaling to GTP-binding proteins. This phosphorylation is reversible, suggesting roles in neuronal functions like neurotransmitter release.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Cell Signaling

Background:

  • Low molecular weight GTP-binding proteins, such as Rap-1b, are crucial in neuronal function.
  • Neuronal signaling pathways involving Ca2+ play vital roles in brain activity.

Purpose of the Study:

  • To investigate the specific interactions between CaM kinase Gr and Rap-1b.
  • To elucidate the role of CaM kinase Gr in the phosphorylation of Rap-1b and its potential implications in neuronal signaling.

Main Methods:

  • In vitro phosphorylation assays using purified CaM kinase Gr and Rap-1b.
  • Characterization of the phosphorylation site on Rap-1b.
  • Investigation of the effect of CaM kinase Gr autophosphorylation on Rap-1b phosphorylation rate.
  • Testing the specificity of CaM kinase Gr towards other Ras superfamily proteins.
  • Assessing the reversibility of Rap-1b phosphorylation using a brain phosphoprotein phosphatase.

Main Results:

  • CaM kinase Gr selectively phosphorylates Rap-1b at a serine residue near its carboxyl terminus with a stoichiometry of approximately 1.
  • Both CaM kinase Gr and cAMP-dependent protein kinase phosphorylate Rap-1b, but CaM kinase II does not.
  • Autophosphorylation enhances the rate of Rap-1b phosphorylation by CaM kinase Gr.
  • CaM kinase Gr does not phosphorylate other tested Ras superfamily proteins (Rab-3A, Rap-2b, c-Ha-ras p21).
  • Phosphorylation of Rap-1b is reversible by an endogenous brain phosphoprotein phosphatase.

Conclusions:

  • CaM kinase Gr is a key enzyme in the phosphorylation of Rap-1b, a brain-enriched GTP-binding protein.
  • This phosphorylation event establishes a link between neuronal Ca2+ signaling pathways and Rap-1b.
  • The findings suggest that Rap-1b phosphorylation by CaM kinase Gr may regulate critical neuronal processes such as transmembrane signaling, vesicle transport, and neurotransmitter release.

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