Comparative analyses of the three-dimensional structures and enzymatic properties of alpha, beta, gamma and delta isoforms of Ca2+-calmodulin-dependent protein kinase II.
Ca(2+)-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (alpha, beta, gamma, and delta) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 A revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K(m) for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca(2+)-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (gamma > beta > delta > alpha) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (beta > gamma > delta > alpha). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.
Gaertner
Tara R
TR
Department of Neurobiology and Anatomy, University of Texas Medical School, 6431 Fannin Street, Houston, TX 77030, USA.
Kolodziej
Steven J
SJ
Wang
Dan
D
Kobayashi
Ryuji
R
Koomen
John M
JM
Stoops
James K
JK
Waxham
M Neal
MN
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry; D020134:Catalytic Domain; D002460:Cell Line; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007313:Insecta; D007700:Kinetics; D008027:Light; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D051381:Rats; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D002147:Calmodulin; D003432:Cross-Linking Reagents; D010455:Peptides; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium