In vitro and in situ phosphorylation of a new endogenous substrate for Ca2+/calmodulin-dependent protein kinase II in bovine adrenal medullary cells.

Shenmai injection (SMI) has been widely used for the treatment of cardiovascular diseases in China. Cardiovascular disorders are often related to excessive catecholamine (CA) secretion. Here, we report the effects of SMI on CA secretion and synthesis in cultured bovine adrenal medullary cells. We found that SMI significantly reduced CA secretion induced by 300 μM acetylcholine (ACh). Cotreatment with SMI (10 μL/mL) and either of the ACh receptor α-subunit inhibitors, HEX (α3) or DhβE (α4β2), did not produce any further inhibition, indicating that SMI may play a role through α3 and α4β2 channels. Furthermore, SMI reduced tyrosine hydroxylase (TH) activity induced by ACh by inhibiting the phosphorylation of TH at Ser19 and Ser40. TH is phosphorylated at Ser19 by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and at Ser40 by protein kinase A (PKA). KN-93 and H89, the antagonists of CaM kinase II and PKA, respectively, inhibited the ACh-induced phosphorylation at Ser19 and Ser40, and the addition of SMI did not augment the inhibitory effect. Taken together, our results show that SMI likely inhibits CA secretion by blocking TH activity at its Ser19 and Ser40 sites.

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In situ hybridization of calcium/calmodulin dependent protein kinase II and tau mRNAs: species differences and relative preservation in Alzheimer's disease

Molecular Brain Research ◽

10.1016/0169-328x(92)90071-i ◽

1992 ◽

Vol 12 (1-3) ◽

pp. 85-94 ◽

Cited By ~ 27

Author(s):

V.H. Mah ◽

T.A. Eskin ◽

A.M. Kazee ◽

L. Lapham ◽

G.A. Higgins

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

In Situ Hybridization ◽

Protein Kinase ◽

Species Differences ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Protein Kinase Ii ◽

Dependent Protein

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Calponin phosphorylation in vitro and in intact muscle

Biochemical Journal ◽

10.1042/bj2960827 ◽

1993 ◽

Vol 296 (3) ◽

pp. 827-836 ◽

Cited By ~ 65

Author(s):

S J Winder ◽

B G Allen ◽

E D Fraser ◽

H M Kang ◽

G J Kargacin ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Gel Electrophoresis ◽

Dependent Protein Kinase ◽

Kinase C ◽

Sds Page ◽

Protein Kinase Ii ◽

Dependent Protein

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.

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Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II

Biochemical Journal ◽

10.1042/bj20071195 ◽

2008 ◽

Vol 412 (2) ◽

pp. 223-231 ◽

Cited By ~ 36

Author(s):

Tao Song ◽

Naoya Hatano ◽

Toshie Kambe ◽

Yoshiaki Miyamoto ◽

Hideshi Ihara ◽

...

Keyword(s):

Protein Kinase ◽

Hippocampal Slices ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Gh3 Cells ◽

Ionophore A23187 ◽

Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein

The mechanisms of NO inhibition of CaMK [Ca2+/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser847 was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser847, which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys6 was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys6 as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys6 may contribute to NO-induced neurotoxicity in the brain.

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