A novel cAMP-stimulated pathway in protein phosphatase 2A activation.
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 Ca(2+) 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.
Feschenko
Marina S
MS
Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA. feschenk@helix.mgh.harvard.edu
Stevenson
Elizabeth
E
Nairn
Angus C
AC
Sweadner
Kathleen J
KJ
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007150:Immunohistochemistry; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054648:Protein Phosphatase 2; D051381:Rats; D016160:Retinoblastoma Protein / Q000378:metabolism
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D020652:Peptide Elongation Factor 2; D016160:Retinoblastoma Protein; C059041:calyculin A; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2