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The PP2A-Associated Protein α4 Is an Essential Inhibitor of Apoptosis

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Science  22 Oct 2004:
Vol. 306, Issue 5696, pp. 695-698
DOI: 10.1126/science.1100537

Abstract

Despite evidence that protein kinases are regulators of apoptosis, a specific role for phosphatases in regulating cell survival has not been established. Here we show that α4, a noncatalytic subunit of protein phosphatase 2A (PP2A), is required to repress apoptosis in murine cells. α4 is a nonredundant regulator of the dephosphorylation of the transcription factors c-Jun and p53. As a result of α4 deletion, multiple proapoptotic genes were transcribed. Either inhibition of new protein synthesis or Bcl-xL overexpression suppressed apoptosis initiated by α4 deletion. Thus, mammalian cell viability depends on repression of transcription-initiated apoptosis mediated by a component of PP2A.

The α4 protein was initially identified as a component of receptor signal transduction complexes in mammalian B and T lymphocytes (1, 2) and was later determined to be broadly expressed (1, 3). It interacts with the catalytic subunit of protein phosphatase PP2A (PP2Ac) as well as those of PP4 and PP6 (4, 5). Binding of α4 to PP2Ac displaces PP2Ac from a dimeric regulatory complex composed of the core A subunit and any of more than 12 variable B components (6). Interaction of α4 with PP2Ac both enhances PP2Ac catalytic activity and alters its substrate specificity (4, 7). Its yeast homolog, Tap42, is a PP2A regulatory subunit that functions in TOR-dependent nutrient sensing (8). In mammalian cells, the association of α4 with PP2Ac is regulated by growth factor signals and modulators of the TOR pathway such as rapamycin (4, 7) (Fig. 1A). However, rapamycin potentiates apoptosis in growth factor–deprived cells (9), so it is difficult to determine whether the decline in association of α4 with PP2Ac contributes to the cellular response to such treatments or occurs as a consequence of the decrease in cell viability.

Fig. 1.

Conditional deletion of the PP2A-associated protein α4 in thymocytes. (A) Association of α4 with PP2Ac is inhibited by rapamycin. The lymphoid progenitor cell line FL5.12 was cultured overnight in growth factor–deficient medium in the presence (+) or absence (–) of 20 nM rapamycin (Rapa) as indicated. Cell lysates were immunoprecipitated (IP) with antibody to PP2Ac followed by immunoblotting with antibodies to α4 or PP2Ac. As a control, an equivalent amount of lysate was immunoblotted with antibody to α4 to detect total α4. (B) Generation of an α4 allele containing loxP sites. Upper panel: Genomic organization of the α4 gene locus. R, Eco RI; B, Bam HI; K, Kpn I; X, Xba I. The bar indicates the probe used for the Southern blots. Lower panel: Targeting construct and different Eco RI fragments generated from different genotypes (wt, α4 wild-type genotype; fl, floxed α4 allele; ko, Cre-deleted α4 allele). (C) Thymocyte cell number was determined in α4wt/Lck-Cre male (wt-Cre) α4wt/fl/Lck-Cre female (wt/fl-Cre), and α4fl/Lck-Cre male (fl-Cre) mice. Values are means ± SD of three mice. (D) Southern blots of DNA prepared from tail (germline), thymus, and purified splenic T cells of different genotypes as described in (C).

To determine whether α4 contributes to the regulation of cell survival, we generated mice deficient in α4. Two constructs were created that each deleted exon I and adjacent sequences of the α4 gene. These were introduced into an embryonic stem (ES) cell line, but a homologous recombinant was not recovered in either case. Because the α4 gene is located on the X chromosome, this result raised the possibility that α4 was an essential gene in the male ES cells. Hence, we prepared a construct containing the α4 gene in which exons III to V were flanked by loxP (Fig. 1B). After electroporation, 4 of 192 clones showed homologous recombination. Introduction of recombinant Cre into these clones failed to yield ES cell clones carrying a deleted α4 gene (10). Next, we generated mice carrying a germline-transmitted α4-floxed allele (α4fl) integrated by homologous recombination. These mice were bred to Lck-Cre transgenic mice to determine the effect of α4 deletion on developing T cells, a nonessential lineage. In α4fl/Lck-Cre male mice, the thymi were depleted of developing T cells (Fig. 1C) and the residual cells were enriched in immature thymocytes (10). Although the Cre-deleted form of the α4 allele was present in the residual thymocytes, these cells died in the thymus, as no T cells with a deleted α4 allele appeared in the periphery (Fig. 1D). Thus, α4 is required for either T cell development or survival.

In female heterozygotes carrying one wild-type and one α4fl allele, thymocyte numbers were reduced by about 60% relative to wild-type mice, but peripheral T cell numbers were normal (Fig. 1C) (10). Most peripheral T cells in female heterozygotes carried a Cre-deleted form of α4fl (Fig. 1D). Because T cell precursors have undergone random X-chromosome inactivation, this finding suggests that the decreased number of thymocytes resulted from the death of cells in which α4 was deleted on the active X chromosome.

To further analyze the consequences of α4 deletion, we generated immortalized mouse embryonic fibroblasts (MEFs) from male α4fl embryos and compared them to littermate α4wt MEFs (fig. S1) (11). Retroviruses encoding either green fluorescent protein (GFP) expressed from an internal ribosome entry site (IRES) alone (vector) or both a Cre recombinase and an IRES-GFP (Cre) were introduced into α4fl or α4wt MEFs, and GFP-positive cells were isolated. Immunoblot analysis of lysates from GFP-positive cells 48 hours and 72 hours after Cre infection showed a decrease and absence of α4 protein, respectively, in α4fl MEFs but not in α4wt MEFs (Fig. 2A). Cell death was observed beginning 48 hours after Cre infection in the α4fl MEFs, and nearly all cells were dead by 120 hours after infection (Fig. 2B). In contrast, the viability of α4wt cells infected with Cre or α4fl infected with vector was not affected (Fig. 2B). Reconstitution of α4 into α4fl cells by stable transfection rescued cell death in response to Cre infection (10). The dying α4-deleted cells displayed the typical features of apoptosis, including cleavage of caspase-3 and cleavage of the caspase substrate PARP [poly(ADP-ribose) polymerase] (Fig. 2C).

Fig. 2.

α4 deletion induces cell death in MEFs. (A) Depletion of α4 protein in α4fl male MEFs after Cre introduction. α4wt (wt) or α4fl (fl) MEFs were infected with MIGR1-GFP-Cre (Cre), and the resulting GFP-positive cells were isolated and analyzed at the time points indicated. Immunoblotting was performed with antibodies to α4 or actin. (B) The percentage of dead cells was determined by the ratio of 4′,6′-diamidino-2-phenylindole (DAPI)–positive cells to GFP-positive cells isolated after infection of α4fl MEFs with MIGR1-GFP-Cre (fl-Cre) or MIGR1-GFP (fl-Vec) or after infection of α4wt MEFs with MIGR1-GFP-Cre (wt-Cre) or MIGR1-GFP (wt-Vec). (C) After 48 hours of Cre infection, cells were fixed and the presence of cleaved caspase-3 was determined with a specific antibody (red). GFP expression of the cells in the same field was visualized with a fluorescein isothiocyanate (FITC) filter (green) and overlapped with DAPI staining (blue). Lower panel: Cell lysates were analyzed by immunoblotting with a PARP-specific antibody.

Apoptosis can be initiated either through biochemical modulation of existing apoptotic regulatory proteins or through transcription and translation–dependent changes in apoptotic regulatory proteins (1214). To distinguish between these two possibilities, we investigated the effect of protein synthesis inhibition on α4 deletion–induced cell death. Addition of the protein synthesis inhibitor cycloheximide (CHX) 48 hours after Cre infection rescued α4fl cells from apoptosis, despite a decrease of α4 protein expression in the presence or absence of CHX (Fig. 3A) (fig. S2).

Fig. 3.

p53 and c-Jun are phosphorylated after α4 deletion. (A) Cycloheximide treatment rescues α4-deleted cells from death. α4fl MEFs were infected with MIGR1-GFP (Vec) or MIGR1-GFP-Cre (Cre). Duplicate cultures were prepared, and after 48 hours of retroviral infection, CHX was added to one of the paired samples. The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. (B) α4fl (fl) or α4wt (wt) MEFs were infected with MIGR1-GFP (–) or MIGR1-GFP-Cre (+). GFP-positive cells were sorted by flow cytometry after 24 hours of infection and collected at 72 hours after retroviral infection. Immunoblotting was performed with different antibodies as indicated. (C) α4fl (fl) MEFs were infected with MIGR1-GFP (Vec) or MIGR1-GFP-Cre (Cre). After 72 hours, cells were fixed and c-Jun phosphorylation was visualized with an antibody to phosphorylated c-Jun Ser63 (red). GFP expression of the cells in the same field was visualized with a FITC filter (green) and overlapped with DAPI staining (blue). (D) α4fl MEFs were infected with MIGR1-GFP-Cre (Cre), and GFP-positive cells were isolated and analyzed at 48 and 72 hours after Cre introduction. Immunoblotting was performed with antibodies as indicated (11). (E) α4fl MEFs were infected with adeno-LacZ (LacZ) or adeno-E6 (E6) followed by either MIGR1-GFP (Vec) or MIGR1-GFP-Cre (Cre) and cell death was quantitated over time. (F) α4fl MEFs were stably transfected with pBabe-Bcl-xL (Bcl-xL) or pBabe (pBabe) retrovirus vector. The selected clones were infected with either MIGR1-GFP (Vec) or MIGR1-GFP-Cre (Cre) and cell death was quantitated over time (means ± SD).

The transcription factor c-Jun is a PP2A substrate and has been implicated in transcription-dependent apoptotic death in response to diverse cellular stresses, including ultraviolet irradiation, heat and osmotic shock, and growth factor withdrawal (15). Its activation involves phosphorylation followed by nuclear translocation. At 72 hours after Cre-mediated α4 deletion, c-Jun phosphorylation on Ser63 increased (Fig. 3B) and accumulated in the nucleus (Fig. 3C). However, no changes in the expression or activation status of the Jun kinases were detected, as measured by their phosphorylation status (Fig. 3B).

To examine the transcriptional changes that occur after α4 deletion, we performed RNA microarray analysis with RNA from α4fl MEFs 48 hours after infection with Cre or vector. Isolated RNA was hybridized to Affymetrix mouse expression microarrays containing more than 39,000 transcripts and variants. Pairwise analysis of the hybridization profiles revealed that among the 20 genes whose expression was most highly increased in the Cre-infected sample (relative to the vector-infected control), six were established p53-dependent targets: p21, Noxa, MDM2, cyclin G, Stk11, and SIP (table S1). Several genes implicated in the intrinsic mitochondrial apoptotic pathway were also induced, including those encoding mDAP-3, Siva, and endonuclease G (10). The up-regulation of p53-dependent transcripts was associated with the accumulation of p53 that was phosphorylated on Ser18 as α4 expression declined (Fig. 3D). In addition to p53 Ser18 phosphorylation, α4-depleted cells accumulated p21 and Noxa proteins.

Because induction of p53 activity is a potent inducer of apoptosis, we made α4fl cells deficient in p53 by stimulating the ubiquitin-dependent proteolysis of p53 (16). Expression of a papilloma virus E6 protein repressed p53 expression (fig. S3) and partially inhibited apoptosis in response to α4 loss (Fig. 3E). Like the proapoptotic p53 gene targets induced in α4-deleted cells, the other proapoptotic genes transcriptionally induced in α4-deleted cells also regulate the intrinsic apoptotic pathway. Overexpression of Bcl-xL, an inhibitor of the intrinsic apoptotic pathway, protected α4fl-deleted cells from cell death; this result indicates that the transcriptional initiation of apoptosis repressed by α4 is mediated through the intrinsic apoptotic pathway (Fig. 3F).

To determine whether the requirement for α4 is restricted to developing and proliferating cells, we assessed the effect of α4 deletion on differentiated adipocytes. Expression of PPARγ (peroxisome proliferator-activated receptorγ), a nuclear hormone receptor that is critical for adipogenesis (17), caused the α4fl MEFs to differentiate into adipocytes, as confirmed by the intracellular accumulation of lipid droplets (Fig. 4A) and by lipid staining with Oil Red O (10). Cells were then infected at high multiplicity with either an adenovirus encoding Cre recombinase or a control adenovirus. Seven days after infection, fewer adipocytes were observed among the Cre-infected cells, most of which were dead or dying (Fig. 4A) (fig. S4). This Cre-induced death resulted in caspase-3 activation (fig. S4). As in proliferating cells, increased phosphorylation of p53 and c-Jun was detected in response to deletion of α4 (Fig. 4A).

Fig. 4.

α4 deletion induces cell death in nonproliferating tissues. (A) Adipocytes generated from α4fl MEFs (fl) as described in (11) were infected with adeno-LacZ (Vec) or adeno-Cre (Cre). Seven days after infection, cells were stained with propidium iodide (PI) and photographed with a tetramethyl rhodamine isothiocyanate (TRITC) filter or bright field (left panel) or analyzed for alterations in protein expression or phosphorylation by immunoblotting with the indicated antibodies (right panel). (B) α4wt (wt) or α4fl (fl) mice were injected with adeno-Cre (Cre). After 6 days, the mice were killed, livers were removed, and liver sections were analyzed by hematoxylin and eosin (H/E) or TUNEL staining. Solid arrowheads indicate apoptotic cells; open arrowheads indicate macrophages with ingested apoptotic cells (left panel). Immunoblotting was performed with liver lysates from α4wt (wt) or α4fl (fl) mice after infection with adeno-Cre (Cre) using antibodies as indicated (right panel).

Next, we assessed the role of α4 in maintaining the viability of differentiated cells in vivo. Three adult α4fl mice and three α4wt mice were injected with an adenovirus encoding Cre through the tail vein, a technique that selectively infects the liver parenchyma (18). At day 5 after injection, all α4fl mice showed signs of illness, with ruffled fur, hunched posture, and rapid breathing. Over the next 24 hours, their condition deteriorated while the α4wt mice remained healthy. All six mice were killed and their livers removed for histological and biochemical analysis (Fig. 4B). Immunoblot analysis of liver lysates revealed that α4 was absent in the α4fl mice and present in the α4wt mice. The lysates from the α4-depleted liver revealed increased phosphorylation of p53 and c-Jun and induction of p21 expression. Liver sections from Cre-infected α4fl mice revealed multiple apoptotic cells, as determined by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. In contrast, TUNEL analysis revealed an absence of apoptotic cells in Cre-infected α4wt mice.

It is surprising that the deletion of a single PP2A regulatory subunit could have such a profound phenotype. Previous studies of α4 deletion in lymphocytes had concluded that α4 is not an essential gene because mature lymphocytes were observed (19, 20). However, our data show that the peripheral T cells that arose in α4fl/Lck-Cre mice failed to delete the α4fl allele, a possibility not addressed in the prior work. The α4 homolog in yeast, Tap42, plays an essential role in suppressing stress response genes in yeast by either repressing PP2Ac/Sit4 activity or altering their substrate specificity (21). Similarly, α4 appears to suppress the stress response factor c-Jun by maintaining it in a more dephosphorylated state. However, because yeast lack both p53 and an apoptotic response, α4 has also been evolutionarily adapted to repress p53-dependent transcription and apoptosis. It is likely that, in addition to its effect on c-Jun and p53, α4 deletion alters the phosphorylation status of more specialized substrates such as Mid1, a protein involved in midline pattern formation that is also a substrate of the α4/PP2Ac complex (22). Moreover, PP2Ac may play an additional role in apoptosis through interaction with other regulatory subunits (23). Nonetheless, the observation that α4 deletion leads rapidly to apoptosis in all cell types tested demonstrates that specific phosphatase complexes play nonredundant and essential roles in the regulation of transcription-induced apoptosis. The data support the hypothesis that in animal cells, apoptosis is a default cell fate (24). In the absence of specific and regulated inhibition, cells initiate new transcription and translation to actively initiate their apoptotic demise.

Supporting Online Material

www.sciencemag.org/cgi/content/full/306/5696/695/DC1

Materials and Methods

Figs. S1 to S4

Table S1

References

References and Notes

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