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

Protein phosphatase 1 (PP1) plays important roles in many different aspects of cellular activities including cell cycle control. One important function of PP1 is to activate the retinoblastoma protein pRB. Here we show that pRB is one of PP1's downstream targets during apoptosis. When HL-60 cells synchronized at the G1/S boundary were treated with pro-apoptotic cytosine arabinoside (araC), PP1α protein increased twofold and PP1 activity about 30% within 1 h. This was followed by pRB dephosphorylation, pRB cleavage by caspases, DNA fragmentation, the appearance of cells with <2n DNA content and finally, dying and dead cells. In vitro, pRB was protected from caspase-3 digestion by prior Cdk-mediated phosphorylation, whereas PP1α converted phospho-pRB into an efficient substrate for caspase-3. Introduction of active PP1α into HL-60 cells by electroporation was sufficient to induce characteristics of apoptosis. Similarly, araC-resistant cells, normally unable to die in response to araC, initiated apoptosis when electroporated with active PP1α. This was also accompanied by pRB cleavage. In contrast, introduction of inhibitor-2 delayed the onset of araC-induced apoptosis, whereas concomitant introduction of PP1α and inhibitor-2 completely prevented PP1α-induced apoptosis. These results suggest that dephosphorylation of key proteins by PP1α may be crucial for the initiation of apoptosis and further support the concept of PP1 serving as a potential target for anti-cancer therapy.