Abstract
Elevated cAMP in NRK-52E and L6 cells causes a marked reduction in the phosphorylation of numerous phosphoproteins, as detected initially with phosphoserine-specific antibodies. Here, we show that elevation of cAMP in NRK cells by forskolin/3-isobutyl-1-methylxanthine (IBMX) treatment decreased phosphorylation of substrates for different protein kinases, pointing to a common protein phosphatase as a target for cAMP-dependent regulation. Forskolin/IBMX treatment completely dephosphorylated a selective protein phosphatase 2A (PP2A) substrate, elongation factor-2 (EF-2), at its Ca2+ calmodulin-dependent kinase site, and decreased phosphorylation of substrates for cyclin-dependent kinases, including retinoblastoma (Rb) protein. As reported before, forskolin/IBMX also decreased phosphorylation of a protein kinase C substrate, the Na,K-ATPase. The cAMP-stimulated dephosphorylation was blocked by the protein phosphatases 1 (PP1) and PP2A inhibitor okadaic acid at concentrations selective for PP2A but was not blocked by tautomycin at concentrations selective for PP1. The data implicate PP2A as a cAMP-activated phosphatase. Contrary to expectation, we found evidence that cAMP-dependent activation of PP2A did not depend on protein kinase A (PKA). Pretreatment of cells with the PKA inhibitor H89 abolished PKA activity measured in cell extracts and significantly decreased cAMP-activated phosphorylation of a known PKA substrate, ARPP-19, in cells, but failed to block the cAMP-stimulated dephosphorylation of EF-2, Rb, and other proteins. This novel pathway of PP2A activation, acting on the time scale of minutes, represents yet another example of a cAMP-mediated, PKA-independent signaling mechanism. Because PP2A is active toward a variety of endogenous substrates, cAMP-stimulated dephosphorylation may have complicated the interpretation of many prior studies.
Footnotes
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This work was supported by National Institutes of Health Grant NS27653 (to K.J.S.), by American Cancer Association Grant IRG-173-J (to M.S.F.), and by National Institutes of Health Grant GM50402 (to A.C.N.).
- Abbreviations:
- PKA
- cAMP-dependent protein kinase
- PKC
- protein kinase C
- PP1 and PP2A
- protein phosphatases 1 and 2A
- cdk
- cyclin-dependent kinase
- EF-2
- elongation factor 2
- Rb
- retinoblastoma protein
- IBMX
- 3-isobutyl-1-methylxanthine
- DMEM
- Dulbecco's modified Eagle's medium
- cAMP-GEF
- cyclic nucleotide-regulated guanine exchange factors
- Received January 28, 2002.
- Accepted March 25, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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