Autophosphorylation of type II CaM kinase in hippocampal neurons

Localization of phosphoand dephosphokinase with complementary phosphorylation site-specific antibodies

Bruce Patton, Sean S. Molloy, Mary B. Kennedy

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

We have visualized the distribution of autophosphorylated type II CaM kinase in neural tissue with the use of two complementary antibodies: a monoclonal antibody that binds to the α and β subunits of the kinase only when they are autophosphorylated at threonine-286 (287 in β) and affinity-purified rabbit antibodies that bind to both subunits only when they are not phosphorylated at these residues. We used these antibodies to double-label organotypic hippocampal cultures, detecting the mouse monoclonal antibody with rhodamine and the rabbit polyclonal antibodies with fluorescein. In double-exposed photographs, the ratios of intensities of the two fluorophores revealed the relative proportion of autophosphorylated and nonphosphorylated kinase in individual neurons throughout the cultures. We found that autophosphorylated and nonphosphorylated kinase are colocalized throughout most neurons rather than segregated within distinct cells or subcellular domains. However, the variations in intensity of the two fluorophores indicated that the proportion of autophosphorylated kinase is consistently higher in neuronal somas than in the neuropil. Incubation of the cultures in Ca2+ free medium dramatically reduced both the level of autophosphorylated kinase detected biochemically and the relative intensity of fluorescent staining with the phosphokinase specific monoclonal antibody. These results support the hypothesis that regulation of Ca2+-independent CaM kinase activity in vivo occurs by a dynamic equilibrium between autophosphorylation and dephosphorylation and that this equilibrium is maintained, at varying steady-state levels, in all parts of neurons.

Original languageEnglish (US)
Pages (from-to)159-172
Number of pages14
JournalMolecular Biology of the Cell
Volume4
Issue number2
StatePublished - 1993
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Phosphotransferases
Phosphorylation
Neurons
Antibodies
Monoclonal Antibodies
Rabbits
Rhodamines
Neuropil
Carisoprodol
Threonine
Fluorescein
Staining and Labeling

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Autophosphorylation of type II CaM kinase in hippocampal neurons : Localization of phosphoand dephosphokinase with complementary phosphorylation site-specific antibodies. / Patton, Bruce; Molloy, Sean S.; Kennedy, Mary B.

In: Molecular Biology of the Cell, Vol. 4, No. 2, 1993, p. 159-172.

Research output: Contribution to journalArticle

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