A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases.
To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.
Kameshita
Isamu
I
Department of Life Sciences, Kagawa University, Kagawa 761-0795, Japan. kamesita@ag.kagawa-u.ac.jp
Tsuge
Toshiyuki
T
Kinashi
Tomoko
T
Kinoshita
Shun
S
Sueyoshi
Noriyuki
N
Ishida
Atsuhiko
A
Taketani
Shigeru
S
Shigeri
Yasushi
Y
Tatsu
Yoshiro
Y
Yumoto
Noboru
N
Okazaki
Katsuichiro
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western / Q000379:methods; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000276:immunology; D018076:DNA, Complementary; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis / Q000276:immunology; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011494:Protein Kinases / Q000032:analysis* / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517685:Camk4 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases