Patton, Bruce L. and Molloy, Sean S. and Kennedy, Mary B. (1993) Autophosphorylation of type II CaM kinase in hippocampal neurons: localization of phospho- and dephosphokinase with complementary phosphorylation site-specific antibodies. Molecular Biology of the Cell, 4 (2). pp. 159-172. ISSN 1059-1524 http://resolver.caltech.edu/CaltechAUTHORS:PATmbc93
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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 alpha and beta subunits of the kinase only when they are autophosphorylated at threonine-286 (287 in beta) 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 Ca(2+)-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.
|Additional Information:||Copyright © 1993 by The American Society for Cell Biology Submitted October 15, 1992; Accepted December 9, 1992 We thank Susan Ou and Julianna Hom-Krist of the Caltech Monoclonal Antibody Facility for their assistance and advice in producing monoclonal antibodies, and Leslie Schenker for expert technical assistance. Peptide synthesis was performed by Lisa L. Chang and Suzanna J. Horvath of the Caltech Biopolymer Synthesis Center. The pcDL-SRa2 expression vector and mutant a subunit constructs were gifts from Robert Bulleit and Masamichi Ito. Financial support was provided by a grant from the Beckman Institute at Caltech, by NIH grant NS-17660 to M.B.K. and training grant NS07251, the Irvine Foundation, the McKnight Foundation, the Gustavus and Louis Pfeiffer Foundation, and the Joseph W. Drown Foundation. B.L.P. was supported in part by a Lawrence L. Ferguson Fellowship at Caltech.|
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