PKCα

In a previous paper (Saito et al., 1998), we reported that PKCα was suggested to be the mediator of the effect of phorbol ester on hGH-BP release and that a PKC-specific inhibitor, Bisindolylmaleimide III, inhibited the PKCα (IC50=0.26±0.05 μM) much more specifically than the other PKCs expressed in IM-9 cells, i.e. PKCδ (IC50=5.7±0.5 μM) and μ(IC50=6.0±0.2 μM). Therefore, we subsequently investigated the effect of bisindolylmaleimide III on the PDBu-enhanced phosphorylation of ecto-proteins (Fig. 6). Phosphorylation of several proteins induced by PDBu-stimulation was dose-dependently inhibited by bisindolylmaleimide III treatment. The IC50 values (n=3) of the phosphorylated bands (42, 45, 53, and 83 kDa) ranged from 0.23 to 0.32 μM, which are similar to that (0.33±0.03 μM) of the PDBu-enhanced hGH-BP release by bisindolylmaleimide III (Saito et al., 1998). This finding suggests that activation of PKCα by PDBu is necessary for the phosphorylation of several ecto-proteins, including 42 (corresponding to 43 kDa protein in Fig. 5), 45, 53, and 83 kDa proteins as well as enhanced hGH-BP release [2].

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Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome [1].

Ecto-kinase assay [2]
IM-9 cells (2×106 cells/ml, 1.5 ml) were washed twice with RPMI 1640 containing 10 mM HEPES–NaOH (pH 7.4) and then suspended in RPMI1640 medium containing 0.6 mM CaCl2, 0.6 mM MgCl2, and 2.5 mM NaF. IM-9 cells were preincubated with or without K-252b or Bisindolylmaleimide III for 15 min at 37°C. The phosphorylation reaction was initiated by addition of [γ-32P]-ATP (74 GBq/mmol; 5 μM in final) with or without 100 nM PDBu. The cells were incubated for 30 min at 37°C, and following addition of 1 mM ATP (final concentration) and 0.25 mg BSA, the reaction was terminated with 0.5 M HClO4 (final concentration). The acid-insoluble cell fractions were washed three times with 0.5 M HClO4 and then three times with ethanol/ether (3/1), as described previously (Amano et al., 1984). The insoluble fractions were solubilized with Laemmli buffer (Laemmli, 1970). The phosphorylated proteins (from 4.5–6×105 cells) were applied to SDS-PAGE followed by analysis with BAS1500.

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Ki Determination of BisIII for the Human Oxidoreductase NQO2 [1]
Enzyme activity and inhibition were determined spectrophotometrically by measuring the reduction of MTT at 610 nm and 30 °C. In this assay, we used NADH as an electron donor for the menadion reduction and MTT for the continuous re-oxidation of menadiol. The reactions (200 μl) were performed in 96-well plates, containing 50 mm KxHxPO4, pH 7.5, 1 μl of quinone-reductase type 2 (NQO2), 40 μm menadion, 200 μm MTT, and increasing concentrations of NADH (0–1000 μm) in the presence of different fixed BisIII concentrations (0, 1, 30, 60 μm). Using a Lineweaver-Burk application, we determined the apparent Km (Km,app) values for the different BisIII concentrations. The Ki was calculated by plotting the different Km,app against its corresponding BisIII concentrations.

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In Vitro Kinase Assays [1]
Lysates from COS-7 cells transiently expressing full-length SLK fused to a N-terminal FLAG-tag were subjected to immunoprecipitation with protein G Sepharose-bound anti-FLAG antibodies for 3 h at 4 °C. After binding, the beads were washed three times with 500 μl of TL-buffer and once with 500 μl of kinase buffer (20 mm HEPES, pH 7.5, 15 mm MgCl2, 80 mm KCl, 1 mm Na3VO4, and 0.1 mm DTT). SLK kinase activity assays were performed in a total volume of 60 μl. Immunoprecipitated SLK bound to 15 μl of drained beads was mixed with 35 μl of kinase buffer containing the indicated BisIII concentrations and incubated for 10 min at 4 °C. The kinase reactions were started by addition of 100 μm ATP, 1 μCi [γ-32P]ATP, and 20 μg of myelin basic protein and incubated for 10 min at 30 °C. CDK2 assays were performed in a total volume of 50 μl. Then 100 ng of CDK2/CyclinA and BisIII, BisVIII, and BisX concentration as indicated were pre-incubated in kinase buffer for 10 min at 4 °C. The kinase reactions were started by adding 100 μm ATP, 2 μCi [γ-32P]ATP, and 10 μg of histone H1 and incubated for 20 min at 30 °C. All kinase reactions were stopped by adding 25 μl of 3× SDS sample buffer. Samples were analyzed by SDS electrophoresis and autoradiography using phosphoimaging for quantification. IC50 calculations were performed with GraFit 5.0

Normalization of the BisIII, BisVIII, and BisX Concentrations [1]
All three bisindolylmaleimide compounds were dissolved in 100% dimethylsulfoxide. Stocks were spectrophotometrically normalized (Spectramax Plus 384; Molecular Devices, Sunnyvale, CA) using their absorption maxima at 372 nm and 460 nm and stored under argon at −20 °C in the dark.

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Immobilization of Bisindolylmaleimides [1]
For immobilization, drained epoxy-activated Sepharose 6B was resuspended in 2 volumes of 20 mm BisIII, BisVIII, or BisX dissolved in 50% dimethylformamide/0.1 M Na2CO3 pH 11. After adding of 10 mm NaOH, coupling was performed overnight at 30 °C in the dark. After three washes with 50% dimethylformamide/0.1 M Na2CO3, remaining reactive groups were blocked with 1 m ethanolamine, pH 11. Subsequent washing steps were performed according to the manufacturer's instructions. To generate the control matrix (Ctrl), epoxy-activated Sepharose 6B was directly reacted with 1 m ethanolamine pH 11 and equally treated as described above. The matrices were stored at 4 °C in the dark.

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Cell Lysis and in Vitro Association Experiments [1]
COS-7 and HeLa cells were usually lysed in Triton X-100 lysis buffer (TL-buffer) containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 1 mm EDTA, 1 mm EGTA, 3 mm CaCl2 plus additives (10 mm sodium fluoride, 1 mm orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 0.2 mm dithiothreitol) and co-factors (100 μg/ml phosphatidylserine (PS), 20 μg/ml DAG)). For analytical in vitro association experiments, lysates were precleared by centrifugation and equilibrated to 1 m NaCl. Then 300 μl of the high salt lysate were incubated together with 20 μl of drained bisindolylmaleimide matrix (Bis matrix) for 3 h at 4 °C. Afterward, the Bis matrices were washed twice with 500 μl of TL-buffer plus 1 m NaCl and once with 500 μl of TL-buffer; both steps were performed without additives, and the co-factor concentrations were reduced to one-tenth. In the experiments designed to detect specific SLK and AK binding to BisIII beads, CaCl2 and the co-factors were not included in the lysis and wash buffers. When the stimulation-dependent binding of Rsk1 to BisIII beads was analyzed, CaCl2 and the co-factors were omitted from the lysis and wash buffers and the NaCl concentration was kept at 150 mm. For activation-dependent detection of PKCα binding from HuH-7 cells, the lysis buffer was 20 mm HEPES, pH 7.5, 150 mm NaCl, 0.25% Triton X-100, 0.1 mm EDTA, 0.2 mm EGTA plus additives. Where indicated, 0.5 mm CaCl2 plus co-factors were added. Co-factor concentrations were also reduced to one-tenth in the respective washing buffer, which was lysis buffer without additives. Bound proteins were eluted by boiling of the affinity beads in 1.5x SDS sample buffer. After SDS-PAGE, proteins were transferred to nitrocellulose membrane and immunoblotted with the corresponding antibodies. The preparative binding experiments using 2.5 × 109 cells, 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE separation and mass spectrometric analysis were carried out essentially as described, with the modification that 3 mm CaCl2, 100 μg/ml PS, and 20 μg/ml DAG were included and glycerol was omitted from the lysis buffer. Moreover, the BisIII column was washed with buffer containing 3 mm CaCl2, 20 μg/ml PS, and 4 μg/ml DAG after sample loading.

[1]. Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors. Mol Cell Proteomics. 2004 May;3(5):490-500.

[2]. Role of ecto-kinase in phorbol ester-enhanced growth hormone-binding protein release from human IM-9 cells. Mol Cell Endocrinol. 1999 Jun 25;152(1-2):65-72.

Bisindolylmaleimide III is a member of maleimides and a member of indoles. It has a role as an EC 2.7.11.13 (protein kinase C) inhibitor. It is functionally related to a maleimide.

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In this work, we have used total cell extracts from HeLa cells as a protein source for the identification of bisindolylmaleimide-binding proteins. Importantly, all bisindolylmaleimide compounds used for immobilization are commercially available reagents. Thus, our established purification protocol can be easily applied to characterize PKC inhibitor-targeted subproteomes in other cell lines, tissue extracts, or even whole organisms and thereby provide a comprehensive picture how this widely used class of signal transduction inhibitors exerts its effects on the molecular level.[1]

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Previously we reported that a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), increased the release of human growth hormone-binding protein (hGH-BP) in IM-9 cells, and that this phorbol ester-enhanced release was mediated by protein kinase Ca (PKCalpha). In the present study, the mechanisms of the phorbol ester-enhanced hGH-BP release were further investigated. Treatment of IM-9 cells with PDBu did not increase hGH-BPs (55-60 kDa) in the intracellular soluble fraction. When the cells were treated with trypsin to remove human growth hormone receptors (hGHRs) on the cell surface after stimulation, no hGH-BPs were detected in the culture supernatants, nor did treatment with bafilomycin A1 or chloroquine affect the PDBu-enhanced hGH-BP release. These results suggest that hGH-BPs released by PDBu stimulation are derived from cell surface hGHRs and not generated within the cells. Protein kinase inhibitors with broad specificities, K-252a and K-252b, inhibited the PDBu-enhanced release with almost the same dose-dependency, although only a trace amount of K-252b was incorporated into IM-9 cells than K-252a, suggesting that K-252b probably inhibits an ecto-kinase extracellularly. PDBu actually enhanced the phosphorylation of several extracellular proteins, and this enhanced phosphorylation was completely inhibited by K-252b treatment. Moreover, the PKCalpha-specific inhibitor Bisindolylmaleimide III which inhibits PDBu enhanced hGH-BP release inhibited the PDBu-enhanced phosphorylation of extracellular proteins. On the other hand, the impermeable PKC inhibitor PKC inhibitor peptide 19-31 did not inhibit PDBu-enhanced release, suggesting that the target PKCalpha for PDBu is not present on the extracellular surface. Taken together, these results suggest that, in addition to intracellular PKCalpha, activation of an undefined ecto-kinase may also be involved in the PDBu-enhanced hGH-BP release. [2]

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C23H20N4O2

384.430504798889

384.158

137592-43-9

2398

Brown to reddish brown solid powder

1.4±0.1 g/cm3

708.7±60.0 °C at 760 mmHg

382.4±32.9 °C

0.0±2.3 mmHg at 25°C

1.739

3.15

3

3

5

29

705

0

O=C1C(=C(C(N1)=O)C1=CNC2C=CC=CC1=2)C1=CN(CCCN)C2C=CC=CC1=2

APYXQTXFRIDSGE-UHFFFAOYSA-N

InChI=1S/C23H20N4O2/c24-10-5-11-27-13-17(15-7-2-4-9-19(15)27)21-20(22(28)26-23(21)29)16-12-25-18-8-3-1-6-14(16)18/h1-4,6-9,12-13,25H,5,10-11,24H2,(H,26,28,29)

3-[1-(3-aminopropyl)indol-3-yl]-4-(1H-indol-3-yl)pyrrole-2,5-dione

3-[1-(3-AMINOPROPYL)-1H-INDOL-3-YL]-4-(1H-INDOL-3-YL)-1H-PYRROLE-2,5-DIONE; 3-(1-(3-Aminopropyl)-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; bisindolylmaleimide iii; 137592-43-9; CHEBI:41059; 3-[1-(3-aminopropyl)indol-3-yl]-4-(1H-indol-3-yl)pyrrole-2,5-dione; Bisindolylmaleimide III, Hydrochloride; BIM III;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 25 mg/mL (65.03 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.25 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.25 mg/mL (3.25 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)