Antibody specific for the Thr-286-autophosphorylated alpha subunit of Ca2+/calmodulin-dependent protein kinase II.
We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KII) autophosphorylated only at Thr-286 of the alpha subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys-289 of alpha subunit of CaM-KII)] was synthesized and phosphorylated by the CaM-KII endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase alpha subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KII both in purified and synaptic cytoskeleton-associated form. Appearance of CaM-KII subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca(2+)-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr-286- or Thr-287-autophosphorylated form of CaM-KII in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KII.
Suzuki
T
T
Department of Biochemistry, Nagoya City University Medical School, Japan.
Okumura-Noji
K
K
Ogura
A
A
Kudo
Y
Y
Tanaka
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003599:Cytoskeleton / Q000378:metabolism; D006624:Hippocampus / Q000201:enzymology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000276:immunology*; D010769:Phosphothreonine / Q000276:immunology; D011494:Protein Kinases / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D013569:Synapses / Q000378:metabolism
D010455:Peptides; D010750:Phosphoproteins; D010769:Phosphothreonine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases