Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin‐dependent protein kinase II self‐association

Calmodulin (CaM)‐kinase II holoenzymes composed of either α or β subunits were analyzed using light scattering to determine a mechanism for self‐association. Under identical reaction conditions, only αCaM‐kinase II holoenzymes self‐associated. Self‐association was detected at a remarkably low enzyme...

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Bibliographic Details
Published in:Journal of neurochemistry 2001-03, Vol.76 (5), p.1364-1375
Main Authors: Hudmon, Andy, Kim, Sally A., Kolb, Stephen J., Stoops, James K., Waxham, M. Neal
Format: Article
Language:English
Subjects:
Amino Acid Sequence
autoinhibition
autophosphorylation
Binding Sites
Biological and medical sciences
calcium
Calcium - metabolism
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases - chemistry
Calcium-Calmodulin-Dependent Protein Kinases - metabolism
Calcium-Calmodulin-Dependent Protein Kinases - ultrastructure
Calmodulin - metabolism
calmodulin‐dependent protein kinase II
Catalytic Domain
Cell physiology
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
ischemia
Kinetics
Light
Microscopy, Electron
Models, Molecular
Molecular and cellular biology
Peptides - chemistry
Peptides - metabolism
Protein Binding
Protein Conformation
Scattering, Radiation
self‐association
Signal transduction
translocation
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Summary:Calmodulin (CaM)‐kinase II holoenzymes composed of either α or β subunits were analyzed using light scattering to determine a mechanism for self‐association. Under identical reaction conditions, only αCaM‐kinase II holoenzymes self‐associated. Self‐association was detected at a remarkably low enzyme concentration (0.14 µm or 7 µg/mL). Light scattering revealed two phases of self‐association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2–3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self‐association required activation by Ca2+/CaM and was strongly dependent on pH. Self‐association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide‐2) and inhibitor (AIP) designed from the autoregulatory domain of CaM‐kinase II potently prevented self‐association, whereas the peptide substrate syntide‐2 did not. Thus, CaM‐kinase II self‐association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory‐like sequence. A model for CaM‐kinase II self‐association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit–interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM‐kinase II previously reported in models of ischemia.
ISSN:0022-3042
1471-4159
DOI:10.1046/j.1471-4159.2001.00119.x