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

Andy Hudmon, Sally A. Kim, Stephen J. Kolb, James K. Stoops, M. Neal Waxham

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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 auto-regulatory 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 auto-regulatory-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.

Original languageEnglish (US)
Pages (from-to)1364-1375
Number of pages12
JournalJournal of Neurochemistry
Volume76
Issue number5
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases
Light transmission
Holoenzymes
Transmission Electron Microscopy
Light scattering
Association reactions
Transmission electron microscopy
Light
syntide-2
Peptides
Catalytic Domain
Calmodulin
Chemical activation
Electron Microscopy
Protein Isoforms
Ischemia
Substrates
Electron microscopy
Enzymes

Keywords

  • Autoinhibition
  • Autophosphorylation
  • Calcium/calmodulin-dependent protein kinase II
  • Ischemia
  • Self-association
  • Translocation

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association. / Hudmon, Andy; Kim, Sally A.; Kolb, Stephen J.; Stoops, James K.; Neal Waxham, M.

In: Journal of Neurochemistry, Vol. 76, No. 5, 2001, p. 1364-1375.

Research output: Contribution to journalArticle

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abstract = "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 auto-regulatory 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 auto-regulatory-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.",
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AU - Stoops, James K.

AU - Neal Waxham, M.

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N2 - 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 auto-regulatory 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 auto-regulatory-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.

AB - 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 auto-regulatory 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 auto-regulatory-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.

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