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 mech anisms by dephosphorylating activated components. By utilizing selecti ve inhibitors of these phosphatases, we investigated their role in reg ulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell li ne. PTHrP, PTH and PGE(2) stimulated cAMP accumulation up to 100-fold. Calyculin A, a potent inhibitor of protein phosphatase type 1 (PPI) a nd type 2A (PP2A), did not affect basal levels of cAMP, but concentrat ions of 10(-11) M to 10(-8) M increased PTHrP-, PTH- and PGE(2)-stimul ated cAMP accumulation up to 1.7-fold, and this increase was concentra tion-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 PGE(2)-stimulated cAMP accumulation. The effect of ca lyculin A on agonist-stimulated cAMP accumulation persisted in cells t reated with isobutyl methylxanthine, a phosphodiesterase inhibitor. Wh en 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 bo th the receptor and forskolin-stimulated cAMP accumulation, calyculin A had no effect on the forskolin-stimulated cAMP accumulation. The eff ect of calyculin A on PTHrP- and PTH-stimulated cAMP accumulation pers isted in cells treated with PMA. These results suggest that protein ph osphatases play an important role in agonist-stimulated cAMP accumulat ion in osteoblastlike cells, and that PPI but not PP2A may tie the maj or phosphatase involved. In contrast to activation by protein kinase C , the site of action for the phosphatase appears to be predominantly a t a step prior to the activation of adenylyl cyclase in the cAMP signa l transduction pathway.