Inhibition of protein kinase C by calphostin C is light-dependent

Bruns, Robert F.; Miller, Freda D.; Merriman, Ronald L.; Howbert, J. Jeffry; Heath, William F.; Kobayashi, Eiji; Takahashi, Isami; Tamaoki, Tatsuya; Nakano, Hirofumi

doi:10.1016/0006-291x(91)90922-t

Cited by 359 publications

(191 citation statements)

References 9 publications

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“…Error bars represent deviation from the average. Calphostin C was activated, after addition to synaptosomes, by exposure to fluorescence lighting for 30 min at room temperature (46). ever, did not lead to a significant reduction in in vitro phosphorylation (data not shown)).…”

Section: Depolarization-dependent Phosphorylation Of Nsf Inmentioning

confidence: 87%

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

Matveeva¹,

Whiteheart²,

Vanaman³

et al. 2001

Journal of Biological Chemistry

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Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. In this report, we demonstrate the depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF. The molecular mechanisms of neurotransmitter (NT)1 release have been the subject of much attention in recent years resulting in an evolving model known as the SNARE hypothesis (1-3). The SNARE hypothesis holds that membrane proteins in the vesicle (v-SNAREs, e.g. synaptobrevins) bind to a heterodimer in the target membrane (t-SNAREs, heterodimers of syntaxins and SNAP-25-like proteins). v-and t-SNAREs bind to form a 7 S complex (1, 4), composed of a bundle of four parallel, coiled-coil domains (5-8) that, through reconstitution studies, has been demonstrated to be minimally required for bilayer fusion (9). The N-ethylmaleimide-sensitive factor (NSF) and the soluble NSF attachment proteins (SNAPs, not to be confused with SNAP-25) affect the composition and structure of the SNARE complex. SNAPs act as adapters and are required for binding of NSF to the 7 S complex. ATP hydrolysis by NSF causes the resulting 20 S complex to disassemble into monomeric SNAREs (4, 10), a step required for vesicle trafficking (11-13). Detailed kinetic experiments have placed one role of NSF and SNAPs at steps prior to v-/t-SNARE binding (14 -16), suggesting a model in which NSF disassembles cis 7 S complexes that exist in the same bilayer (17-19). The resulting monomeric SNAREs can then form trans 7 S complexes that span the opposing bilayers of the vesicle and target membrane. In this manner, NSF acts as a chaperone to activate or "prime" the SNARE proteins for subsequent trans complex formation and membrane fusion.Studies by Schweizer et al. (20), using NSF-derived peptides microinjected pre-synaptically, showed that ...

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Section: Depolarization-dependent Phosphorylation Of Nsf Inmentioning

confidence: 87%

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

Matveeva¹,

Whiteheart²,

Vanaman³

et al. 2001

Journal of Biological Chemistry

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“…Particularly noteworthy is the increased inhibitory potency of RO 31 8220 and staurosporine against membrane-derived as compared to cytosolic enzyme, and the markedly decreased ability of CGP 41251 and calphostin C to inhibit membranederived in comparison to cytosolic PKC. Calphostin C does not only inhibit PKC, but also inactivates it irreversibly in a lightdependent fashion [17]. Gopalakrishna et al showed that calphostin C exhibited higher potency against membrane-derived than cytosolic PKC in experiments in which cells were treated with the drug preceeding preparation of cellular subfractions and subsequent determination of enzyme activity [18].…”

Section: Discussionmentioning

confidence: 99%

Differential inhibition of cytosolic and membrane‐derived protein kinase C activity by staurosporine and other kinase inhibitors

Budworth

Gescher

1995

FEBS Letters

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The hypothesis was tested that 9 kinase inhibitors with diverse specificities for protein kinase C (PKC), including staurosporine and four of its analogues, interfere differently with PKC derived from either the cytosolic or particulate fractions of MCF-7 breast carcinoma cells. GF 109203X inhibited the enzyme identically in either preparation. CGP 41251 and calphostin C inhibited cytosolic PKC more effectively than membrane-derived PKC with ratios of ICso (cytosolic PKC) over IC50 (membrane-derived PKC) of 0.07 and 0.04, respectively. The other six agents inhibited membrane-derived PKC more potently than cytosolic enzyme. Staurosporine and RO 31 8220 exhibited IC50 ratios of 12.3 and 21.6, respectively. The results suggest that there are dramatic differences between kinase inhibitors in their divergent effects on cytosolic and membrane-derived PKC which should be borne in mind in the interpretation of their pharmacological properties.but possess much better selectivity for PKC [7][8][9][10]. Two such PKC-specific analogues, UCN-01 and CGP 41251, have been shown to possess antineoplastic activity in human tumour models grown in rodents [9,11]. In most investigations of the ability of compounds to inhibit PKC, cytosolic enzyme preparations have been used. In the light of the fact that it is predominantly PKC in the membrane which is of functional importance, we tested the hypothesis that kinase inhibitors display different potencies against membrane-derived as compared to cytosolic enzyme. To that end nine well-characterised PKC inhibitors, including staurosporine and four analogues, were investigated with respect to their ability to inhibit PKC prepared from the cytosol or membrane of human-derived MCF-7 breast cancer cells. The susceptibility of these cells towards the growth-arresting ability of PKC inhibitors has recently been described [12].

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“…Thus, intracellular calcium, calcium store-operated calcium channels, and TRP channels are not significantly involved. Application of the protein kinase C inhibitor, 0.5 !M calphostin (> 20 min) under normal fluorescent lighting condition (Bruns et al, 1991) could not abolish the carbachol effect (1 mM carbachol, n = 5, vehicle water only, n = 3; frequencies increased by 2.3 ± 1.9 vs. 0.4 ± 1.1 Hz, P = 0.005, U-test).…”

Section: Inhibition Of Intracellurar Pathways Such As Plc or Inositolmentioning

confidence: 93%

Muscarinic receptor type 1 (M1) stimulation, probably through KCNQ/Kv7 channel closure, increases spontaneous GABA release at the dendrodendritic synapse in the mouse accessory olfactory bulb

Takahashi

Kaba

2010

Brain Research

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Cholinergic modulation of spontaneous GABAergic currents (mIPSC) was studied using whole-cell patch methods in mouse accessory olfactory bulb slices. Carbachol (above 100 !M) administration produced an increase in the mIPSC frequency in mitral cells, but did not affect the responses of mitral cells to GABA. The carbachol effect persisted in the presence of combined ionotropic and metabotropic glutamatergic receptor antagonists. The carbachol effect was reduced by the muscarinic receptor type-1 and -4 (M1, M4) antagonist pirenzepine (10 !M), but not by the M2 and M4 antagonist himbacine (10 !M). The KCNQ/Kv7 potassium channel openers retigabine (80 !M) and diclofenac (300 !M) blocked the carbachol action, while the KCNQ potassium channel blocker XE-911 (20 !M) increased the mIPSC frequency. XE-911's action persisted in the presence of glutamate receptor blockers. In the presence of carbachol, mIPSCs were abolished by Ni (200 !M), while being insensitive to the calcium channel blocker nimodipine (30 !M), suggesting a role for R-type calcium channels in the GABA release. These results suggest that carbachol closed KCNQ channels by stimulating M1 receptors on granule cell dendrites, and the resulting depolarized and unstable membrane promoted calcium influx, thus increasing the GABA release. The possible role of acetylcholine in facilitating formation of a pheromone memory in mice is also discussed. Classification TermsSection: Neurophysiology

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Inhibition of protein kinase C by calphostin C is light-dependent

Cited by 359 publications

References 9 publications

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

Differential inhibition of cytosolic and membrane‐derived protein kinase C activity by staurosporine and other kinase inhibitors

Muscarinic receptor type 1 (M1) stimulation, probably through KCNQ/Kv7 channel closure, increases spontaneous GABA release at the dendrodendritic synapse in the mouse accessory olfactory bulb

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