Protein phosphatase 1alpha-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), PP1alpha 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 PP1alpha converted phospho-pRB into an efficient substrate for caspase-3. Introduction of active PP1alpha 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 PP1alpha. This was also accompanied by pRB cleavage. In contrast, introduction of inhibitor-2 delayed the onset of araC-induced apoptosis, whereas concomitant introduction of PP1alpha and inhibitor-2 completely prevented PP1alpha-induced apoptosis. These results suggest that dephosphorylation of key proteins by PP1alpha may be crucial for the initiation of apoptosis and further support the concept of PP1 serving as a potential target for anti-cancer therapy.
Wang
R H
RH
Division of Hematology/Oncology, Childrens Hospital Los Angeles, University of Southern California School of Medicine, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA.
Liu
C W
CW
Avramis
V I
VI
Berndt
N
N
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003561:Cytarabine / Q000494:pharmacology; D053938:DNA Fragmentation; D005434:Flow Cytometry; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D000964:Antimetabolites, Antineoplastic; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D003561:Cytarabine; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases