kn-62 and Pheochromocytoma

Involvement of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) on the phosphorylation of tyrosine hydroxylase (TH, EC.1.14.16.2) in rat pheochromocytoma, PC12h cells was examined using KN-62, 1-[N,O-Bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipe razine, a selective inhibitor of Ca2+/CaM-kinase II. Both the enhanced phosphorylation of TH and the activated L-3,4-dihydroxyphenylalanine (DOPA) formation in the high K+ depolarization were inhibited by 10 microM KN-62. After incubation of PC12h cells with 10 microM KN-62 for 1 hr, the activation of TH with 3 min incubation of 56 mM K+ was reduced to the basal activity. However, KN-62 did not directly affect the activity of purified rat TH at pH 6.0 or 7.0. These results indicate that Ca2+/CaM-kinase II phosphorylates and activates TH of PC12h cells in the high K+ depolarization.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenal Gland Neoplasms; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Dihydroxyphenylalanine; Enzyme Activation; Isoquinolines; Kinetics; Molecular Weight; Pheochromocytoma; Phosphorylation; Piperazines; Potassium; Protein Kinase Inhibitors; Rats; Tyrosine 3-Monooxygenase

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Binding Sites; Binding, Competitive; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Chickens; Chromatography, Affinity; Cyclic AMP; Gizzard, Avian; Isoquinolines; Kinetics; Myosin-Light-Chain Kinase; Myosins; Pheochromocytoma; Phosphorylation; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Rats; Tumor Cells, Cultured

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenal Gland Neoplasms; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin-Binding Proteins; Cell Differentiation; Isoquinolines; Neoplasm Proteins; Pheochromocytoma; Phosphorylation; Piperazines; Protein Binding; Protein Kinase Inhibitors; Protein Kinases; Protein Processing, Post-Translational; Rats; Second Messenger Systems; Substrate Specificity; Sulfonamides; Tumor Cells, Cultured