Kinase assay

Total kinase activity of CaMKII, determined in a standard 2 min assay (100 μL), contained 35 mM HEPES, 10 mM MgCl2, 1 mM CaCl2, 10 μg of chicken gizzard myosin 20-kD light chain, 0.1 μM calmodulin and 10 μM [γ-33]ATP at 30 ℃. The kinase reaction was halted by adding 1 mL of 10% trichloroacetic acid.

Cell lines

K562 cells

Preparation method

The solubility of this compound in DMSO is > 36.1 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

1, 2, 5 or 10 μM; 24 or 48 hrs

Applications

KN-62 inhibited the cell growth of K562 cells in a dose-dependent manner. Two days after the treatment of 10 μM KN-62, 63% K562 cells were inhibited. Flow cytometric analysis showed that KN-62 (10 μM, 24 hrs) caused an accumulation of K562 cells in S phase. Immunoblotting studies indicated that CaMKII was inhibited in these K562 cells.

KN-62, 1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-_L-tyrosy]-4-phenylpiperazine is a highly selective inhibitor of the calcium dependent calmodulin protein kinase II (CAMKII), which binds to the calmodulin binding site of CAMKII and consequently does not inhibit other classes of calmodulin-sensitive kinases. In previous studies, KN-62 exhibits inhibitory effects on the regulated secretion of insulin by hetero-junction with intrinsic thin-layer (HIT) cells and the cholecystokinin secretion from stanniocalcin-1 (STC-1) enteroendocrine cells due to a block of Ca²⁺influx through L-type Ca²⁺channels in the olasma membrane. It also inhibits both insulin- and hypoxia-stimulated glucose transport activity by 46% and 40% respectively in skeletal muscle.

Reference

[1].Joseph T. Brozinick, Jr., Thomas H. Reynolds, David Dean, Gregory Cartee and Samuel W. Cushman. 1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosy]-4-phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle. Biochem. J. (1999) 339, 533-540
[2].Erik S. Schweitzer, Michael J. Sanderson and C. G. Wasterlain. Inhibition of regulated catecholamine secretion from PC12 cells by the Ca2+/calmodulin kinase II inhibitor KN-62. Journal of Cell Science 108. 2619-2628 (1995)