Inhibition of serine/threonine protein phosphatases enhances agonist-stimulated cAMP accumulation in UMR 106 osteoblast-like cells.

Protein phosphatases regulate the activity of signal transduction mechanisms by dephosphorylating activated components. By utilizing selective inhibitors of these phosphatases, we investigated their role in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line. PTHrP, PTH and PGE2 stimulated cAMP accumulation up to 100-fold. Calyculin A, a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A (PP2A), did not affect basal levels of cAMP, but concentrations of 10(-11) M to 10(-8) M increased PTHrP-, PTH-, and PGE2-stimulated cAMP accumulation up to 1.7-fold, and this increase was concentration-dependent. Similar results were obtained with tautomycin, another potent inhibitor of PP1 and PP2A. In contrast, okadaic acid, a potent inhibitor of PP2A which inhibited PP1 less potently, did not enhance PTHrP-, PTH-, or PGE2-stimulated cAMP accumulation. The effect of calyculin A on agonist-stimulated cAMP accumulation persisted in cells treated with isobutyl methylxanthine, a phosphodiesterase inhibitor. When the effect of calyculin A was compared with that of 4 beta-phorbol 12-myristate 13-acetate (PMA), it was found that while PMA enhanced both the receptor and forskolin-stimulated cAMP accumulation, calyculin A had no effect on the forskolin-stimulated cAMP accumulation. The effect of calyculin A on PTHrP- and PTH-stimulated cAMP accumulation persisted in cells treated with PMA. These results suggest that protein phosphatases play an important role in agonist-stimulated cAMP accumulation in osteoblast-like cells, and that PP1 but not PP2A may be the major phosphatase involved. In contrast to activation by protein kinase C, the site of action for the phosphatase appears to be predominantly at a step prior to the activation of adenylyl cyclase in the cAMP signal transduction pathway.