title: Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.
abstract: While kinases are typically composed of one or two subunits, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is composed of 8-14 subunits arranged as pairs around a central core. It is not clear if the CaMKII holoenzyme functions as an assembly of independent subunits, as catalytic pairs, or as a single unit. One strategy to address this question is to genetically engineer monomeric and dimeric CaMKII and evaluate how their activity compares to the wild-type (WT) holoenzyme. Here a technique that combines fluorescence correlation spectroscopy and homo-FRET analysis was used to characterize assembly mutants of Venus-tagged CaMKIIα to identify a dimeric CaMKII. Spectroscopy was then used to compare how holoenzyme structure and function changes in response to activation with CaM in the dimeric mutant, WT-holoenzyme, and a monomeric CaMKII oligomerization-domain deletion mutant control. CaM triggered an increase in hydrodynamic volume in both WT and dimeric CaMKII without altering subunit stoichiometry or the net homo-FRET between Venus-tagged catalytic domains. Biochemical analysis revealed that the dimeric mutant also functioned like WT holoenzyme in terms of its kinase activity with an exogenous substrate, and for endogenous T286 autophosphorylation. We conclude that the fundamental functional units of CaMKII holoenzyme are paired catalytic-domains.
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
authors.forename: Pabak
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
authors.forename: Kaitlin A
authors.initials: KA
authors.lastname: Davis
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
authors.forename: Henry L
authors.initials: HL
authors.lastname: Puhl
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
authors.forename: Jithesh V
authors.initials: JV
authors.lastname: Veetil
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
authors.forename: Tuan A
authors.initials: TA
authors.lastname: Nguyen
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland. Electronic address: stevevog@mail.nih.gov.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Vogel
mesh_terms: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D020035:Holoenzymes / Q000737:chemistry; D006801:Humans; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D020035:Holoenzymes; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2