Differential and Selective Inhibition of Protein Kinase A and Protein Kinase C in Intact Cells by Balanol Congeners
- Departments of Pharmacology and Medicine (Å.B.G., L.L.B.) and the Biomedical Sciences Graduate Program (Å.B.G.), University of California at San Diego, La Jolla, California
Abstract
The fungal metabolite balanol is a potent inhibitor of protein kinase A (PKA) and protein kinase C (PKC) in vitro that acts by competing with ATP for binding (Ki ∼ 4 nM); congeners of balanol show specificity for PKA over PKC. We have characterized the effects of balanol and 10"-deoxybalanol in intact cells to determine whether these compounds cross the cell membrane and whether the potency and specificity noted in vitro are preserved in vivo. In neonatal rat myocytes and cultured A431 cells transiently transfected with a cyclic AMP response element-luciferase reporter construct, balanol inhibits the induction of luciferase activity by isoproterenol, indicating inhibition of PKA. Western analysis shows that both balanol and 10"-deoxybalanol reduce phosphorylation of cAMP response element-binding protein in isoproterenol-stimulated A431 cells; inhibition is concentration dependent with an IC50value of ∼3 μM. Balanol, but not 10"-deoxybalanol, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, a PKC substrate, in phorbol ester-stimulated A431 cells (IC50 ∼ 7 μM). Our data demonstrate that balanol is a potent inhibitor of PKA and PKC in several whole-cell systems and causes no obvious toxicity. In addition, balanol congeners inhibit PKA and PKC with the specificity and potency predicted by in vitro experiments.
Footnotes
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Send reprint requests to: Dr. Åsa B. Gustafsson, Department of Pharmacology 0636, University of California San Diego, School of Medicine, La Jolla, CA 92093-0636. E-mail,agustafsson{at}ucsd.edu
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This work was supported by National Institutes of Health Grants HL-41307 and GM-07752.
- Abbreviations:
- cAMP
- cyclic AMP
- PKA
- cyclic AMP-dependent protein kinase
- PKC
- protein kinase C
- MARCKS
- myristoylated alanine-rich C kinase substrate
- CRE
- cyclic AMP response element
- CREB
- cyclic AMP response element-binding protein
- TCA
- trichloroacetic acid
- ATF-1
- activating transcription factor-1
- DMEM
- Dulbecco’s modified Eagle’s medium
- PMA
- phorbol-12-myristate-13-acetate
- PAGE
- polyacrylamide gel electrophoresis
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- Received February 11, 1999.
- Accepted May 8, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
