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Proc Natl Acad Sci U S A. Nov 22, 2011; 108(47): 18937-18942.
Published online Nov 14, 2011. doi:  10.1073/pnas.1110988108
PMCID: PMC3223437

Differential effects on p53-mediated cell cycle arrest vs. apoptosis by p90


p53 functions as a central node for organizing whether the cell responds to stress with apoptosis or cell cycle arrest; however, the molecular events that lead to apoptotic responses are not completely understood. Here, we identified p90 (also called Coiled-Coil Domain Containing 8) as a unique regulator for p53. p90 has no obvious effects on either the levels of p53 or p53-mediated cell cycle arrest but is specifically required for p53-mediated apoptosis upon DNA damage. Notably, p90 is crucial for Tip60-dependent p53 acetylation at Lys120, therefore facilitating activation of the proapoptotic targets. These studies indicate that p90 is a critical cofactor for p53-mediated apoptosis through promoting Tip60-mediated p53 acetylation.

The p53 tumor suppressor acts as the major sensor for a regulatory circuit that monitors signaling pathways from diverse sources, including DNA damage, oncogenic events, and other abnormal cellular processes (1, 2). p53 monitors and responds to a multitude of stress signals by coordinating cell growth arrest or apoptosis (35). Central to p53 regulation of these cellular processes is its activity as a transcription factor, although transcription-independent functions of p53 are also critical under some biological settings. The activation of p53 transcription activity requires multiple steps, including sequence-specific DNA binding, antirepression and acquirement of combinations of posttranslational modifications, and recruitment of corepressors/coactivators in a promoter-specific manner (6). To suppress tumor growth, p53 induces either cell growth arrest or apoptosis depending on the cellular context. The molecular mechanisms that govern the choice between growth arrest and apoptosis are extremely important but not well understood (4, 7). As a key player in the stress response, p53 demands an exquisitely complicated network of control and fine-tuning mechanisms to ensure correct, differentiated responses to the various stress signals encountered by cells (2, 5, 810).

p53 was the first nonhistone protein known to be regulated by acetylation and deacetylation (11, 12). There is accumulating evidence indicating that acetylation of p53 plays a major role in activating p53 function during stress responses (2, 13, 14). Following our early findings of C terminus p53 acetylation, we and others recently showed that p53 is also acetylated by Tip60 (also known as KAT5)/MOF (human ortholog of males absent on the first) at residue Lys120 (K120) within the DNA-binding domain (1517). K120 acetylation is crucial for p53-mediated apoptosis but has no obvious effect on p21 expression, an essential target of p53-mediated growth arrest. Notably, although Tip60 is required for K120 acetylation of p53 in vivo, the levels of K120 acetylation are dynamically regulated in vivo and the interaction between p53 and Tip60 is not very stable, indicating that additional regulators may play a role in controlling K120 acetylation and subsequent p53-mediated apoptotic response (1820). Through biochemical purification, we identified p90 as a unique regulator for p53. p90, also called CCDC8 (coiled-coil domain containing 8), which was previously found down-regulated in human cancer cells (21, 22), interacts with p53 both in vitro and in vivo. Knockdown of p90 has no obvious effect on p53-mediated activation of p21 but specifically abrogates its effect on p53 upregulated modulator of apoptosis, also known as Bbc3 (PUMA) activation. Moreover, p90 also interacts with Tip60 and promotes Tip60-dependent Lys120 acetylation of p53, therefore enhancing the apoptotic response of p53. These data reveal p90 as an upstream regulator of the Tip60-p53 interaction and demonstrate that p90 is specifically required for p53-mediated apoptosis upon DNA damage.


Identification of p90 as a Unique Component of p53-Associated Complexes.

To further elucidate the mechanisms of p53-mediated promoter-specific activation in vivo, we isolated p53-associated protein complexes from human cells. Attempts to purify p53-containing protein complexes were hindered in the past because cells cannot tolerate expressing even low levels of wild-type p53. Interestingly, our recent studies indicate that p538KR, in which all eight p53 acetylation sites are mutated to arginine (Fig. 1A), is completely inactive in inducing cell cycle arrest or apoptosis (23). Moreover, p538KR retains the capacity to bind target gene promoters as well as to activate the p53-Mdm2 (Mouse double minute 2) feedback loop, suggesting that p538KR, unlike the hot spot tumor mutant p53H175R, may retain a similar conformation as wild-type p53 in human cells. Therefore we have utilized an H1299 p53-null lung carcinoma cell line that stably expresses a double-tagged human p538KR mutant protein with N-terminal FLAG and C-terminal HA epitopes (FLAG-p538KR-HA). To ensure physiological interactions, we used H1299 derivatives that express the ectopic p538KR protein at levels comparable to those of endogenous p53 in HCT116 colon cancer cells upon DNA damage treatment. As expected, Mdm2 is activated in the p538KR stable line to a similar level compared to that induced by DNA damage in HCT116 cells. Consistent with previous findings, proapoptotic and growth-arrest targets such as PUMA and p21 are not activated in the p538KR stable line (Fig. 1B). To isolate p538KR-containing complexes, cell extracts from the stable line were subjected to a two-step affinity chromatography previously described (24). The tandem affinity-purified p53-associated proteins were analyzed by liquid chromatography (LC) MS/MS. As expected, we identified known p53 binding proteins such as Mdm2, tumor protein 53 binding protein 1, ubiquitin specific peptidase 7, and the CREB-binding protein as specific components of the p53 complex. In addition, MS analysis of a protein band p90 (with the apparent size at approximately 90 kDa molecular mass) revealed six peptide sequences matched with a signal cDNA sequence in the database, which is also named CCDC8 (Fig. 1C). Because none of the peptide sequences of p90 were identified from the control complexes purified in parental H1299 cells, p90 is likely a unique binding partner of p53. The cDNA of p90/CCDC8 encodes a 538 amino acid protein which possesses no known functional domains other than two small coiled-coil regions that are likely to mediate protein–protein interactions (Fig. 1C). Although p90/CCDC8 has been reported as a candidate tumor suppressor gene in renal cell carcinoma (RCC), the molecular function of this protein is unclear (25).

Fig. 1.
Identification of p90 as a component of a p53-containing protein complex. (A) Schematic representation of the p538KR protein used for protein complex purification. Mutations of acetylation sites are indicated. TAD, transcription activation domain; PRD, ...

p90 is a Bona Fide p53 Interacting Protein.

To investigate a role for p90 in regulating p53 function in vivo, we first tested the interaction between p90 and p53. Thus, we first transfected H1299 cells with expression vectors for FLAG-tagged p53 and HA-tagged p90. Western blot analysis revealed that p90 is readily detected in p53-associated immunoprecipitates (Fig. 2A). Because p90 was identified in the p53 complex purified from cell extracts, we assessed the cellular localization of the p90–p53 interaction. To this end, we established a U2OS cell line stably expressing FLAG and HA double-tagged p90. Using parental U2OS cells as control, we fractionated the cell extracts from the p90 stable line and immunoprecipitated both nuclear and cytoplasmic fractions with M2/FLAG agarose beads. Western blot analysis showed that p53, as expected, localizes mainly in the nuclear fraction, whereas p90 is present in both fractions but more so in the cytoplasmic fraction (Fig. 2B). Furthermore, in the M2 immunoprecipitate, p53 is primarily found in the nuclear fraction (Fig. 2B). The difference in p90/p53 ratio in the cytoplasmic and nuclear fractions as well as the p53 abundance in the nuclear M2 immunoprecipitate indicate that, although p90 is present in both the cytoplasm and the nucleus, it interacts with p53 predominantly in the nucleus.

Fig. 2.
p90 is a bona fide p53 interacting protein. (A) p90 coimmunoprecipitates with p53 in an overexpression system. H1299 cells were transiently transfected with plasmid DNA expressing HA-p90 or/and FLAG-p53. The cell extracts and the M2 immunoprecipitates ...

To further elucidate this interaction under physiological settings, we then raised an affinity-purified polyclonal antiserum against the full-length p90 protein. Upon Western blot analysis, this antibody specifically detected in human cells an approximately 90 kDa polypeptide (Fig. 2C, lane 1), the level of which decreases significantly after treatment with p90-specific siRNA oligos (Fig. 3A). To investigate the interaction between endogenous p90 and p53 proteins, extracts from U2OS osteosarcoma cells were immunoprecipitated with α-p53 or with the control IgG. As expected, the α-p53 antibody immunoprecipitated endogenous p53; more importantly, p90 is easily detected in the immunoprecipitates obtained with the α-p53 antibody but not the control IgG (Fig. 2C, lanes 2 and 3), confirming that p90 and p53 interact endogenously. An in vitro GST-pulldown assay was performed to further assess direct interaction. p53 can be divided into an N-terminal (NT) fragment containing the transactivation domain, a middle fragment (M) containing the DNA-binding domain, and a C-terminal (CT) fragment containing the tetramerization domain as well as the regulatory domain. Purified recombinant GST-tagged p53 full-length and fragment proteins were incubated with in vitro translated 35S-methione-labeled HA-p90. Following immobilization with GST resins and recovery of captured complexes using reduced glutathione, the eluted complexes were resolved by SDS-PAGE and analyzed by autoradiography. 35S-labeled HA-p90 strongly bound immobilized GST-tagged full-length and CT fragment of p53 (Fig. 2D, lanes 2 and 5), but not the NT and middle fragments of p53 or GST alone (Fig. 2D, lanes 3, 4, and 6). These data demonstrate that p90 interacts with p53 in vitro through binding directly to the C-terminal portion of p53.

Fig. 3.
Inactivation of p90 reduces basal PUMA level and differentially affects p53-mediated PUMA and p21 induction upon DNA damage. (A) p90 RNAi does not affect p53 stability or p21 basal level but reduces basal PUMA expression. U2OS cells were transiently transfected ...

Inactivation of p90 Attenuates p53-Mediated Activation of PUMA but Not p21.

To understand the physiological role of p90, we examined whether inactivation of endogenous p90 has any effect on the stability and functions of p53. To this end, U2OS cells were transfected with a p90-specific (p90-RNAi#1) siRNA oligo or a control (control-RNAi) siRNA oligo. As shown in Fig. 3A, lanes 1 and 2, the level of endogenous p90 polypeptides was severely reduced after transfection with p90-RNAi. p53 protein level was unaffected by p90 ablation, suggesting that p90 does not regulate p53 stability. We then assessed the effect of p90 inactivation on the level of two important p53 downstream targets: the growth-arrest target p21 and the apoptotic target PUMA. Surprisingly, p90 ablation displayed differential effects on the two different endogenous targets: The level of PUMA was significantly reduced, whereas p21 expression remained unchanged. To exclude off-target effects, we also treated cells with three additional p90 siRNAs (p90-RNAi#2, p90-RNAi#3; p90-RNAi#4) that recognize different regions of the p90 mRNA. Again, the levels of PUMA were decreased by p90 knockdown, although there was no significant change for the levels of p53 and p21 (Fig. 3A, lanes 3–5). Because p53 is strongly activated upon DNA damage and regulates downstream targets, we wanted to assess whether p90 affects p53 and downstream target activation upon DNA damage. U2OS cells were transfected with p90-specific siRNA oligos followed by treatment with the DNA damage reagent etoposide. As expected, p53 levels increased drastically upon DNA damage (Fig. 3B, lane 2 vs. lane 1), and notably, RNAi-mediated ablation of p90 displays no effect on p53 accumulation following etoposide treatment (Fig. 3B, lane 4, 6, 8, and 10 vs. lane 2). p21 was strongly induced upon treatment, however, damage-induced PUMA expression was severely attenuated in the cells treated with p90-RNAi (Fig. 3B, lanes 4, 6, 8, 10 vs. lane 2).

To validate that the differential effect of p90 on PUMA and p21 is p53 dependent, we inactivated both p53 and p90 in U2OS using RNAi prior to etoposide treatment. In cells transfected with the control siRNA, p53 accumulates and both PUMA and p21 are activated significantly upon treatment (Fig. 3C, lanes 1 and 2). In a p53-deficient background, DNA damage fails to activate PUMA and p21 (lanes 3 and 4), and more importantly p90 ablation displayed no effect on PUMA and p21 in the absence of p53 (lanes 5 and 6). Taken together, these data demonstrate that p90 inactivation differentially affects PUMA and p21 induction in a p53-dependent manner.

p90 is Required for p53-Mediated Apoptosis upon DNA Damage.

To further confirm the differential effects on p53-mediated activation of p21 versus PUMA, we collected the cells at different time points following treatment with etoposide. At all time points, p53 accumulation and p21 activation were unaffected by p90 ablation but PUMA induction was severely attenuated (Fig. 4A, lanes 5 and 6 vs. lanes 2 and 3). We furthered confirmed that p90 ablation affected p53-dependent activation of p21 and PUMA at the transcription level by examining the mRNA levels of these targets. Indeed, basal PUMA mRNA was reduced in samples treated with p90-RNAi, consistent with our finding that p90 ablation reduces basal PUMA protein level (Fig. 3A). PUMA activation was attenuated at the mRNA level following p90 ablation, whereas p21 mRNA level increased upon etoposide treatment at all time points and remained unaffected in samples treated with p90-RNAi. To further confirm these differential effects of p90 in p53 responses, we repeated these experiments in the cells treated with another DNA damage reagent doxirubicin. Again, we observed the differential effects of p90 on p53-dependent p21 and PUMA activation upon doxirubicin treatment (Fig. 4B, lanes 6–8 vs. lanes 2–4). These results suggest that p90 is crucial for p53-dependent activation of PUMA, which is a very important mediator of p53-mediated apoptosis (2628). We therefore speculated whether the attenuation of PUMA activation by loss of p90 can be translated into a phenotypic effect on apoptosis. To this end, we transfected U2OS cells with either control siRNA or p90 siRNA prior to etoposide treatment. Cells were collected at different timepoints, stained with propidium iodide (PI), and analyzed by flow cytometry for apoptotic cells according to DNA content. As shown in Fig. 4C, basal level sub-G1 content is minimally affected by inactivation of p90. However, following 18 or 24 h of etoposide treatment, an average of 18.67% or 26.84% of cells transfected with control siRNA were apoptotic, whereas only 8.04% or 11.49% of cells transfected with p90 siRNA were apoptotic (Fig. 4D). These data demonstrate that p90 is crucial for p53-mediated apoptosis.

Fig. 4.
Inactivation of p90 attenuates p53-dependent PUMA activation in time point experiments and impairs apoptosis upon damage. (A and B) p90 RNAi reduces PUMA but not p21 activation upon DNA damage. U2OS cells transiently transfected with either control siRNA ...

Mechanistic Insights into p90-Mediated Effect on p53-Dependent Apoptotic Responses.

Previous studies demonstrated that p53 acetylation at Lys120 (p53 AcK120) by Tip60 is indispensable for apoptosis but not required for growth arrest (16, 17), leading to our speculation that p90 may regulate p53-mediated PUMA activation through promoting Tip60-dependent acetylation of p53 at K120. To investigate the mechanism underlying the effect of p90 on the apoptotic target PUMA, we first assessed the interaction between p90 and Tip60. To this end, H1299 cells were transfected with expression vectors for Tip60 and FLAG/HA double-tagged p90. Western blot analysis revealed that Tip60 is readily detected in p90 associated immunoprecipitates (Fig. 5A). Using a GST-pulldown assay, we further tested the in vitro interaction of Tip60 and p90. As shown in Fig. 5B, Tip60 bound to immobilized GST-tagged p90 but not GST alone, demonstrating that p90 and Tip60 interacts directly. To investigate the role of p90 in p53 K120 acetylation by Tip60, we examined whether Tip60-mediated p53 acetylation is modulated by p90 status. As expected, Western blot analysis revealed that p53 was readily acetylated by Tip60 (Fig. 5C, lane 2 vs. lane 1). Notably, although p90 itself does not acetylate p53 (Fig. 5C, lane 3), p53 acetylation by Tip60 was significantly enhanced upon p90 expression (Fig. 5C, lane 4 vs. lane 2). These data demonstrate that p90 promotes the aceylation of p53 by Tip60.

Fig. 5.
p90 interacts with Tip60 and promotes Tip60-mediated p53 acetylation at K120. (A) Tip60 coimmunoprecipitates with p90 in an overexpression system. H1299 cells were transiently transfected with the plasmid DNA expressing Tip60 or/and FLAG-HA-p90. Cell ...

In order to confirm the effect of p90 on Tip60-mediated p53 K120 acetylation under physiological settings, we inactivated p90 in U2OS cells via RNAi and assessed endogenous acetylation of p53 at K120. Because the steady-state levels of K120 acetylation are dynamically regulated by both acetylases and deacetylases, in order to exclude the potential effect on p53 acetylation levels by deacetylases, cells were treated with deacetylase inhibitors trichostatin A (for inhibiting histone deacetylase 1/histone deacetylase 2-mediated deacetylation of p53) and nicotinamide (for inhibiting Sirt1-mediated deacetylation of p53) prior to harvesting (16, 18, 19). Cell extracts were immunoprecipitated with α-Ac-p53K120 or control IgG. As shown in Fig. 5D, p53 acetylation at K120 was easily detected in the cells with the deacetylase inhibitor treatment; however, the levels of p53 acetylation at K120 were significantly reduced upon p90 knockdown (Fig. 5D, lane 4 vs. lane 2). Taken together, these data indicate that p90 is a critical cofactor for p53-mediated apoptosis through promoting K120 acetylation of p53.


Our findings reveal that p90 is a p53 interacting protein with differential effects on p53-mediated activation of target genes. Here, we have demonstrated that p90 is a bona fide p53 interacting protein and that this interaction primarily occurs in the nucleus. Inactivation of p90 attenuates apoptosis due to down-regulation of p53-mediated PUMA activation upon DNA damage. However, p90 does not appear to affect growth-arrest targets such as p21. To dissect the molecular mechanism underlying this differential regulation, we found that p90 interacts with the Tip60 acetyltransferase and promotes Tip60-mediated acetylation of p53 at K120, a posttranslational modification that has previously been reported to modulate the decision between cell cycle arrest and apoptosis (1517). Thus, p90 likely serves as an upstream regulator of the p53-Tip60 interplay that is required for apoptotic signaling and allows for transcription induction of PUMA in cells at risk of DNA damage.

K120 is located within the p53 DNA-binding domain and is recurrently mutated in cancer (UMD_TP53 mutation database http://p53.free.fr/). Acetylation at K120 is indispensable for activation of proapoptotic targets but is not required for activation of growth-arrest targets (16, 17). Although the mechanism underlying this target specificity remains to be elucidated, it is possible that acetylation at K120 may impose specificity through altering the p53 quaternary structure and thus endowing p53 binding to low-affinity response elements that are found on proapoptotic promoters (29, 30). We also noticed a small amount of cytoplasmic p53–p90 interaction. Cytoplasmic localization of p53 was originally thought to passively block transactivation in the nucleus. However increasing evidence suggests cytoplasmic p53 has important roles in regulating apoptosis and autophagy. Cytoplasmic p53 promotes apoptosis through increasing mitochondrial outer-membrane permeabilization and release of cytochrome c (3133). Basal levels of wild-type p53 in the cytoplasm also inhibits autophagy, although the exact mechanism remains to be understood (34). It will be interesting to explore the possibilities of p90 regulating transcription-independent functions of p53 in the cytoplasm.

It is noteworthy that p90 itself is underexpressed in human tumors, including kidney cancer and myeloma, based on the cancer gene expression profile database from Oncomine Research (21, 22). In this regard, p90 has also been identified as a candidate tumor suppressor gene as hypermethylation and transcriptional silencing of the p90 promoter was found in 35% of primary RCC tumor samples (25). It will be interesting to test whether p53-mediated apoptosis is abrogated in the human tumors lacking p90 expression and whether reactivation of silenced p90 promotes apoptosis thereby contributing to tumor suppression. Finally, protein modifications of the components in the p53 pathway are well accepted as the key mechanisms for controlling p53 function during stress responses (2, 35). Interestingly, p90 contains two potential ataxia telangiectasia mutated/ataxia telangiectasia and Rad3 related (ATM/ATR) phosphorylation sites at Ser-199 and Ser-302 (Fig. 1C). Indeed, in a screen assay performed by the Elledge Group (36), a phosphorylated peptide derived from p90 was identified as an ATM/ATR substrate. Future investigations are required to dissect whether p90 phosphorylation upon DNA damage modulates its interaction with p53 and Tip60 as well as p53-mediated apoptotic responses. It is possible that p90 is functionally regulated by ATM/ATR mediated phosphorylation during the DNA damage response to control the decision between cell cycle arrest and apoptosis mediated by p53.

Materials and Methods

Plasmids, Antibodies and Cell Culture.

The full-length p90 cDNA was amplified by PCR from Human MGC Verified FL cDNA (Open Biosystems) and subcloned into pcDNA3.1/V5-His-Topo vector (Invitrogen), pCIN4-FLAG-HA, or pCIN4-HA expression vector (37). To construct the GST-p90 plasmid, cDNA sequences corresponding to the full-length p90 were amplified by PCR from other expression vectors and subcloned into pGEX (GST) vectors for expression in bacteria.

The polyclonal antibody specific for p90 was generated by Covance. Rabbits were immunized with purified full-length GST-p90 protein. Antisera from the immunized rabbits were first depleted with a GST-affinity column, then affinity purified by use of a GST-p90 affinity column using the Aminolink Plus Immobilization kit (Thermo Scientific). Antibodies used for Western blot analysis are p53 (DO-1), β-tubulin (D-10), p21 (C-19 and SX118), and proliferating cell nuclear antigen (PC10) from Santa Cruz, β-actin (AC-15), PUMA (NT), and FLAG M2 from Sigma, Mdm2 (Ab-5) from EMD Biosciences, HA (3F10) from Roche Applied Science, and α-Acp53K120 antibody (16). Anti-Tip60 (CLHF) was a gift from Chiara Gorrini and Bruno Amati (European Institute of Oncology, Milan, Italy).

H1299 and U2OS cells were maintained in DMEM (Cellgro) and HCT116 cells in McCoy’s 5A medium (Cellgro). All media were supplemented with 10% fetal bovine serum (Gibco). The stable cell lines were established by transfecting H1299 or U2OS cells with the plasmids pCIN4-FLAG-p538KR-HA and pCIN4-FLAG-HA-p90, respectively, followed by selection with 1 mg/mL or 0.5 mg/mL G418 (EMD Biosciences). Transfections with plasmid DNA were performed using the calcium phosphate method and siRNA transfections by Lipofectamine2000 (Invitrogen) according to the manufacturer’s protocol.

Western Blot Analysis and Immunoprecipitation.

For Western blot analysis, cells were lysed in cold FLAG lysis buffer [50 mM Tris·HCl (pH 7.9), 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 10% glycerol, and freshly supplemented protease inhibitor cocktail]. For immunoprecipitations, cells were lysed in cold BC100 buffer [20 mM Tris, (pH 7.9), 100 mM NaCl, 10% glycerol, 0.2 mM EDTA, 0.2% Triton X-100, and freshly supplemented protease inhibitor]. For immunoprecipitations of ectopically expressed FLAG-tagged proteins or from the U2OS FH-p90 stable line, extracts were incubated with the monoclonal M2/FLAG agarose beads (Sigma) at 4 °C overnight. After five washes with the lysis buffer, the bound proteins were eluted using FLAG-peptide (Sigma) in BC100 for 2 h at 4 °C. The eluted material was resolved by SDS-PAGE and immunoblotted with antibodies as indicated.

Preparation of Cytoplasmic and Nuclear Fractions.

Cytoplasmic extracts were prepared by resuspension of pelleted cells in hypotonic buffer [10 mM Tris (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, supplemented with fresh protease inhibitor] followed by Dounce homogenization (six strokes with Type A pestle) and subsequent low-speed pelleting of nuclei (600 × g for 10 min). The supernatant was removed for use as cytoplasmic extract. The pellet from the low-speed spin was washed once with hypotonic buffer containing 0.1% Nonidet P-40, and further extracted with BC200 [20 mM Tris (pH 7.9), 200 mM NaCl, 10% glycerol, 0.2 mM EDTA, 0.4% Triton X-100], supplemented with fresh protease inhibitor. The nuclear extract was then clarified by high-speed centrifugation (21,885 × g for 15 min). For subsequent immunoprecipitation, both fractions were adjusted to a final concentration of 150 mM NaCl and 0.2% Triton X-100.

GST-Pulldown Assay.

GST and GST-tagged protein fragments were purified as described previously (38). 35S-methione-labeled proteins were prepared by in vitro translation using the TNT Coupled Reticulocyte Lysate System (Promega). GST or GST-tagged fusion proteins were incubated with in vitro translated 35S-methione-labeled proteins overnight at 4 °C in BC100 containing 0.2% Triton X-100 and 0.2% BSA. GST resins (Novagen) were then added, and the solution was incubated at 4 °C for 3 h. After five washes, the bound proteins were eluted for 1.5 h at 4 °C in BC100 containing 0.2% Triton X-100 and 20 mM reduced glutathione (Sigma), and resolved by SDS-PAGE. The presence of 35S-labeled protein was detected by autoradiography.

siRNA-Mediated Ablation of p90 and p53.

Ablation of p90 was performed by transfection of U2OS cells with siRNA duplex oligonucleotides [p90-RNAi-1 (5′-GGACUUGACAACUGACGAA-3′), p90-RNAi-2 (5′-GGCAAGAAGGUGCGCAAAA-3′), p90-RNAi-3 (5′-GCAGAUAAUCAGAGGGCGG-3′), and p90-RNAi-4 (5′-ACACAAUGGGGUUGCGUCA-3′)] synthesized by Dharmacon. Ablation of p53 was performed by transfection of U2OS cells with siRNA duplex oligoset (On-Target-Plus Smartpool L00332900, Dharmacon). Control RNAi (On-Target-Plus siControl nontargeting pool D00181010, Dharmacon) was also used for transfection. RNAi transfections were performed two times with Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen).


This work was supported in part by grants from National Institutes of Health, National Cancer Institute, the Leukemia and Lymphoma Society and The Ellison Medical Foundation. We thank E. McIntush from Bethyl, Inc. for developing the antibodies for p90/CCDC8. W.G. is an Ellison Medical Foundation Senior Scholar in Aging.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database [accession no. JN703457 (p90)].


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