THE INVOLVEMENT OF PROTEIN PHOSPHATASES IN THE ACTIVATION OF ICE CED-3 PROTEASE, INTRACELLULAR ACIDIFICATION, DNA DIGESTION, AND APOPTOSIS/

Many events in apoptosis have been identified but their temporal relat ionships remain obscure, Apoptosis in human ML-1 cells induced by etop oside is characterized by intracellular acidification, enhanced Hoechs t 33342 fluorescence, DNA digestion, chromatin condensation, and prote olysis of poly(ADP-ribose) polymerase, This proteolysis is a marker fo r the action of ICE/CED-3 proteases, which are critical activators of apoptosis, We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, din, prevente d all of these apoptotic characteristics. To determine which protein p hosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phos phatase 1 but not protein phosphatase 2A. Rb was dephosphorylated duri ng apoptosis, and each inhibitor prevented this dephosphorylation at t he same concentrations that prevented apoptosis, No increase in protei n phosphatase 1 activity was observed in apoptotic cells suggesting th at dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity, L ong term inhibition of protein phosphatase 1 (> 8 h) also led to the a ppearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymer ase and apoptosis, suggesting these events are not solely dependent up on protein phosphatase 1, Rb dephosphorylation was also observed in se veral other models of apoptosis, Hence, an imbalance between protein p hosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.