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Protein Phosphatase 1A-Mediated Stimulation of Apoptosis Is Associated with Dephosphorylation of the Retinoblastoma Protein

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Protein Phosphatase 1A-Mediated Stimulation of Apoptosis Is Associated with Dephosphorylation of the Retinoblastoma Protein Oncogene (2001) 20, 6111 ± 6122 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Protein phosphatase 1a-mediated stimulation of apoptosis is associated with dephosphorylation of the retinoblastoma protein Rui-Hong Wang1,2, Cathy WY Liu1, Vassilios I Avramis1 and Norbert Berndt*,1 1Division of Hematology/Oncology, Childrens Hospital Los Angeles, University of Southern California School of Medicine, 4650 Sunset Boulevard, Los Angeles, California, CA 90027, USA Protein phosphatase 1 (PP1) plays important roles in number. Pathways that control these two processes are many dierent aspects of cellular activities including cell often linked to each other. It is thus not surprising that cycle control. One important function of PP1 is to protein phosphatase 1 (PP1), one of the major activate the retinoblastoma protein pRB. Here we show mammalian serine/threonine-speci®c phosphatases ubi- that pRB is one of PP1's downstream targets during quitously expressed, is involved in dierent aspects of apoptosis. When HL-60 cells synchronized at the G1/S cell growth control (Berndt, 1999) and may be a target boundary were treated with pro-apoptotic cytosine for cancer therapy (Berndt, 2000). arabinoside (araC), PP1a protein increased twofold and The assignment of speci®c roles in various cellular PP1 activity about 30% within 1 h. This was followed by processes to PP1 is complicated by the fact that pRB dephosphorylation, pRB cleavage by caspases, mammalian PP1 comprises four isozymes (1a,1d, DNA fragmentation, the appearance of cells with 52n 1g1, 1g2) which can bind to many non-substrate DNA content and ®nally, dying and dead cells. In vitro, cellular proteins functioning as regulatory subunits pRB was protected from caspase-3 digestion by prior (Damer et al., 1998). These subunits modify PP1's Cdk-mediated phosphorylation, whereas PP1a converted activity or target it to particular locales and substrates. phospho-pRB into an ecient substrate for caspase-3. PP1 appears to play several roles in cell cycle Introduction of active PP1a into HL-60 cells by regulation. In organisms from S. pombe to humans, electroporation was sucient to induce characteristics PP1 activity is required for the exit from mitosis of apoptosis. Similarly, araC-resistant cells, normally (Axton et al., 1990; Booher and Beach, 1989; Cheng et unable to die in response to araC, initiated apoptosis al., 2000; Fernandez et al., 1992; Ohkura et al., 1989), when electroporated with active PP1a. This was also although the essential mitotic substrate(s) for PP1 have accompanied by pRB cleavage. In contrast, introduction not yet been identi®ed. Meanwhile we have found that of inhibitor-2 delayed the onset of araC-induced phosphorylation of a C-terminal threonine (Thr320) in apoptosis, whereas concomitant introduction of PP1a mammalian PP1a by Cdks inhibits its activity in vitro and inhibitor-2 completely prevented PP1a-induced (Dohadwala et al., 1994). This site is phosphorylated in apoptosis. These results suggest that dephosphorylation vivo in S. pombe at the onset of mitosis (Yamano et al., of key proteins by PP1a may be crucial for the initiation 1994) and in human cells in mitosis (Kwon et al., 1997; of apoptosis and further support the concept of PP1 Liu et al., 1999) and in late G1 until early S phase (Liu serving as a potential target for anti-cancer therapy. et al., 1999). This indicates that control of PP1 Oncogene (2001) 20, 6111 ± 6122. function by regulatory subunits may not be sucient, but also requires post-translational modi®cation of the Keywords: protein phosphatase 1; retinoblastoma catalytic subunit itself, at least in the context of the cell protein; caspase; protein phosphorylation; cell cycle; cycle. Several laboratories including ours have identi- apoptosis ®ed PP1 to be responsible for the dephosphorylation of pRB during the M/G1 transition (Alberts et al., 1993; Berndt, 1995; Durfee et al., 1993; Ludlow et al., 1993) Introduction and G0/G1 phase (Berndt, 1995). In fact, the inactivation of PP1a by Cdks during the G1/S Reversible protein phosphorylation plays an important transition appears to be linked to the concomitant role in the regulation of the cell cycle and apoptosis, inhibition of pRB: Thus, in human cells expressing which are the two major mechanisms to control the cell intact pRB, constitutively active PP1a (PP1aT320A) prevents pRB phosphorylation and progression into S phase (Berndt et al., 1997). Consistent with this result, G1/S phosphorylation aects only pRB-asso- *Correspondence: N Berndt; E-mail: [email protected] ciated PP1a (Liu et al., 1999). Furthermore, in cells 2 Current address: NIDDK, National Institutes of Health, Building that cannot phosphorylate pRB or do not express 10, Room 9N104, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 6 April 2001; revised 21 June 2001; accepted 28 June pRB, PP1a causes increased cell death, giving rise to 2001 the speculation that it is the phosphorylated form of Roles of PP1 in apoptosis R-H Wang et al 6112 pRB, rather than RB per se, that protects cells from aphidicolin and exposed to 10 mM araC for 2 h. Then apoptosis (Berndt et al., 1997). the cells were incubated for another 24 h in the These ®ndings appear to be in con¯ict with reports absence of the drug. At several intervals, samples were that suggested an inhibitory role for pRB in apoptosis, collected and subjected to Western blot analysis, PP1 a notion that is dicult to reconcile with its established activity assay, DNA fragmentation assay, trypan blue role as a tumor suppressor. Thus, massive cell death staining and ¯ow cytometry (Figure 1). The Western occurs in mice lacking pRB expression (Clarke et al., blots, PP1 activity and DNA fragmentation assays are 1992; Jacks et al., 1992; Lee et al., 1992). Consistent based on equal cell number or equal protein. The data with these data, overexpression of wild-type pRB in show that following araC treatment, PP1a protein pRB-lacking tumor cells inhibits apoptosis (Berry et more than doubled its amount 3 h after release from al., 1996; Fan et al., 1996; Haas-Kogan et al., 1995). araC, whereas of the other PP1 isozymes, PP1d More recently, pRB was found to be cleaved by ICE- increased approximately twofold at 24 h after release like proteases during apoptosis generating a 42-amino- from araC, and PP1g1 remained unaected (Figure acid or 5-kDa peptide from its C terminus (Chen et al., 1a). We also observed that PP1 activity increased by 1997; JaÈ nicke et al., 1996). These data suggest that about 30% (Figure 1b). Meanwhile, high molecular pRB belongs to the so-called death substrates which weight pRB gradually disappeared in favor of a faster- need to be destroyed in order to launch or execute migrating species (Figure 1c), which is indicative of apoptosis. However, since none of the above studies either dephosphorylation or proteolytic cleavage or addressed the role that the pRB phosphorylation status both (Tan and Wang, 1998). The DNA fragmentation may play in apoptosis, we concluded that the exact assay, trypan blue staining and ¯ow cytometry function of pRB in apoptosis remains ill-de®ned. analysis demonstrated that araC indeed induced Similarly, the function of protein phosphatases in apoptosis in G1/S synchronized HL-60 cells: they apoptosis is controversial. Thus, prolonged exposure of showed the typical features of apoptosis ± the appear- cells to phosphatase inhibitors such as okadaic acid has ance of DNA laddering (Figure 1d), cells with 52n been shown to cause cell cycle arrest (SchoÈ nthal and DNA content (Figure 1e), and dying or dead cells Feramisco, 1993; Taylor et al., 2000; Zheng et al., 1991) (Figure 1e). The degree of synchronization and the or apoptosis (Bùe et al., 1991; Inomata et al., 1995; progression through the cell cycle of treated cells is Morimoto et al., 1997). In contrast, apart from our own summarized in Table 1. Considering the succession of observations described above, a positive role for protein events, we notice that PP1 activity started to increase phosphatases in cell death was suggested by earlier within 1 h of araC treatment and reached its peak at ®ndings that apoptosis in human tumor cells is associated 1 h following the termination of exposure to araC. with the dephosphorylation of several proteins (Baxter DNA laddering and apoptotic peaks in ¯ow cyto- and Lavin, 1992) and that protein phosphatase activity is metric analyses appeared at about 2 h after release necessary during early apoptosis in plants (Dunigan and from araC, and ®nally, cells positive for trypan blue Madlener, 1995). Moreover, induction of apoptosis by showed up at 6 h following araC treatment. The cytosine arabinoside (araC) in HL-60 cells leads to observed order of events suggests that during S phase, dephosphorylation of pRB by an unidenti®ed phospha- PP1a, but not other PP1 isozymes, may critically be tase (Dou et al., 1995). In the present study, we sought to involved in launching apoptosis. characterize the role of PP1 in apoptosis, with an It should be noted that etoposide- and actinomycin emphasis on the mechanism of interaction between D-induced apoptosis was also associated with an PP1, pRB and caspases. Using HL-60 leukemia cells treated with araC, we found that PP1 activity rapidly and transiently increased approximately 30%, which was Table 1 Cell cycle progression of HL-60 cells treated with araC for accompanied by a twofold increase in the amount of 2h PP1a protein. This was followed by pRB dephosphor- % of cells ylation and then the proteolytic release of a short peptide Time (h) 52n DNA G1 S G2/M from pRB's C terminus. We also demonstrate that Async 3 38 49 13 phospho-pRB was a poor substrate for caspase-3 in vitro, 0 2 75 15 10 while prior dephosphorylation by PP1a converts phos- 1 1 76 16 8 pho-pRB into an ecient caspase-3 substrate.
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