Article Figures & Data
Figures
- FIG. 1.
Expression of active CaPkc1 kinase in insect cells. (A) Left panel, Coomassie blue-stained gel after electrophoresis of insect cell lysates containing CaPkc1 and CAT. Right panel, Western blot analysis of insect cell lysates as shown in the left panel with an anti-His tag antibody. (B) Left panel, Coomassie blue-stained gel after electrophoresis of the IP complex with the anti-His tag antibody of the insect cell lysates as shown in panel A. Right panel, kinase activity present in the immuno-complex after IP with the anti-His tag antibody of the insect cell lysates. (C) Coomassie blue-stained gel showing purification of the N-terminal His-tagged CaPkc1 protein from insect cell lysates through a Ni2+-charged affinity column with an imidazole gradient. The solid arrowhead indicates the CaPkc1 protein, and the open arrowhead shows the CAT protein.
- FIG. 2.
Regulated expression of CaPkc1 protein in C. albicans under control of the C. albicans GAL1-10 promoter. (A) Upper panel, Northern blot analysis of GAL1 gene expression, showing induction by galactose and repression by glucose in C. albicans. Lower panel, ethidium bromide-stained gel of total RNA used for the Northern blot analysis as shown in the upper panel. An overnight culture of C. albicans was collected by centrifugation and washed two times with a medium containing no carbon source. The collected C. albicans cells were then added into fresh medium containing either glucose or galactose as the sole carbon source. To repress GAL1 gene expression, glucose was directly added to cells growing in the galactose-containing medium. (B) Galactose-induced expression of HA-tagged CaPkc1 (solid arrow) by C. albicans cells. Induction of CaPkc1 expression was done similarly as described for panel A, and expression of the HA-tagged CaPkc1 was detected by Western blotting with an anti-HA tag antibody. (C) Kinase activity of the wt and the constitutively active mutant CaPkc1 (CaPkc1RP). The wt and the mutant CaPkc1 proteins were first isolated by IP with the HA tag antibody, and the immuno-complex was then analyzed by Western blotting (upper panel) and assayed for kinase activity (lower panel). (D) Agar plates of various C. albicans strains growing in glucose- or galactose-containing medium, showing the lethality by overexpression of the mutant CaPkc1RP protein on the galactose-containing medium.
- FIG. 3.
Activation of CaPkc1 kinase activity by Rho1 and PS. (A) Coomassie blue-stained gel showing expression and purification of the GST fusion CaRho1. Lane 1, soluble bacterial lysate before induction with isopropyl-β-d-thiogalactopyranoside (IPTG); lane 2, soluble bacterial lysates after IPTG induction; lane 3, affinity-purified GST-CaRho1(arrow). (B) GTP binding of the purified GST-CaRho1 protein as shown in panel A and the competition assay with cold GTP and other trisphosphate nucleotides. (C) CaPkc1 kinase activity in the presence or absence of CaRho1 and PS as indicated. (D) Dose-dependent activation of CaPkc1 kinase activity by PS. PS and DAG were added immediately after sonication into the kinase assay mixture, right before the start of the kinase reaction.
- FIG. 4.
High-throughput kinase assay development. (A) Time course of the CaPkc1 kinase reaction. Phosphorylation of the biotinylated peptide substrate was determined at 1-h intervals. (B) Evaluation of signal strength of kinase activity readout with various combinations of hot ATP, cold ATP, peptide substrate concentrations, and various amounts of streptavidin-coated SPA beads.
- FIG. 5.
Isolation and determination of cercosporamide as a selective Pkc1 kinase inhibitor. (A) The active compound purified and its structure determined by NMR and MS as cercosporamide, a previously known antifungal natural product with an unknown mode of action. (B and C) Cercosporamide is a highly potent, ATP-competitive Pkc1 kinase inhibitor, with an IC50 of <50 nM and a Ki of <7 nM.
- FIG. 6.
Antifungal activity of cercosporamide is mediated through selective inhibition of Pkc1 kinase activity. (A) Cercosporamide has markedly enhanced antifungal activity in the presence of doxycycline. (B) Suppression of the antifungal activity of cercosporamide by 1 M sorbitol.
- FIG. 7.
Cercosporamide and a β-1,3-glucan synthase inhibitor, an echinocandin analog, together have markedly enhanced antifungal activity. (A) MICs of cercosporamide (Cercos), echinocandin analog (ECB), and amphotericin B (amphoB) against C. albicans. (B) Dramatically increased antifungal activity of cercosporamide, seen as a marked decrease in its MIC in the presence of ECB at an ECB concentration that by itself has no apparent antifungal activity.
Tables
- TABLE 1.
PKCβ kinase inhibitors lack activity against CaPkc1
- TABLE 2.
Cercosporamide is a highly potent and selective CaPkc1 inhibitor
Kinase IC50 (μM) Selectivity ratio vs CaPkc1 CaPkc1 0.044 1 PKCα 1.022 23 PKCβ 0.349 8 PKCγ 5.772 130 PKCε 1.574 36 CDK2/cyclin A 12.548 283 CDK1/cyclin B 5.199 117 CDK4/cyclin D1 >20 >451 CDK2/cyclin E >20 >451 GSK 3β 1.338 30 CAMKII 1.505 34 PKAa >20 >451
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Files in this Data Supplement:
- Legends, figure, table S1 - Legends, Table 1, Fig.3, Sussman et al, 2004. Word document, 157K
- Fig. S1 and S2 - Supplementary figures, Sussman et al, 2004. Powerpoint document, 4.07MB.
