Protein phosphatase 1 alpha-mediated stimulation of apoptosis is associated with dephosphorylation of the retinoblastoma protein

Protein phosphatase I (PPI) plays important roles in many different aspects of cellular activities including cell cycle control. One important functio n of PP1 is to activate the retinoblastoma protein pRB. Here we show that p RB is one of PPI's downstream targets during apoptosis. When HL-60 cells sy nchronized at the G1/S boundary were treated with pro-apoptotic cytosine ar abinoside (araC), PP1 alpha protein increased twofold and PP1 activity abou t 30% within I h. This was followed by pRB dephosphorylation, pRB cleavage by caspases, DNA fragmentation, the appearance of cells with < 2n DNA conte nt and finally, dying and dead cells. In vitro, pRB was protected from casp ase-3 digestion by prior Cdk-mediated phosphorylation, whereas PP1 alpha co nverted phospho-pRB into an efficient substrate for caspase-3. Introduction of active PP1 alpha into HL-60 cells by electroporation was sufficient to induce characteristics of apoptosis. Similarly, araC-resistant cells, norma lly unable to die in response to araC, initiated apoptosis when electropora ted with active PP1 alpha. This was also accompanied by pRB cleavage. In co ntrast, introduction of inhibitor-2 delayed the onset of araC-induced apopt osis, whereas concomitant introduction of PP1 alpha and inhibitor-2 complet ely prevented PP1 alpha -induced apoptosis. These results suggest that deph osphorylation of key proteins by PP1 alpha may be crucial for the initiatio n of apoptosis and further support the concept of PPI serving as a potentia l target for anti-cancer therapy.