Investigation of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II translocated to the postsynaptic density.
To elucidate the physiological significance of the translocation of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), we investigated substrates of CaM kinase II in the postsynaptic density (PSD). PSD proteins were phosphorylated by CaM kinase II of its PSD complex, and separated by two-dimensional gel electrophoresis. More than 28 proteins were phosphorylated under experimental conditions. Proteins corresponding to CaM kinase II substrates were excised from the gels, eluted electrophoretically, and then sequenced. Several substrates were identified, including PSD95, SAP90, alpha-internexin, neurofilament L chain, cAMP phosphodiesterase, and alpha- and beta-tubulin. Some substrates were also identified by immunoblotting, including N-methyl-D-aspartic acid (NMDA) receptor 2B subunit, 1-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor 1 (GluR1), neurofilament H chain and dynamin. PSD95, SAP90, dynamin, and alpha-internexin were demonstrated for the first time to be substrates of CaM kinase II. NMDA receptor 2B subunit and GluR1 existed as major substrates in the PSD. Moreover, translocation of CaM kinase II was inhibited by phosphorylation of PSD proteins. These results suggest that CaM kinase II plays important roles in the regulation of synaptic functions through phosphorylation of PSD proteins.
Yoshimura
Y
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1, Tokushima 770-8505, Japan.
Aoi
C
C
Yamauchi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D016548:Prosencephalon / Q000378:metabolism; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism; D013379:Substrate Specificity; D013569:Synapses / Q000378:metabolism
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D018091:Receptors, AMPA; C501449:Dlg4 protein, rat; C033652:postsynaptic density proteins