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Oncogene (2002) 21, 974 ± 979 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc
Regulation of p73 by c-Abl through the p38 MAP kinase pathway Ricardo Sanchez-Prieto1,2, Victor Javier Sanchez-Arevalo2,3, Joan-Marc Servitja1,3 and J Silvio Gutkind*,1 1
Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, MD 20892-4330, USA; 2Clinica Puerta de Hierro, Departamento de Anatomia Patologica, Unidad de Patologia Molecular, C/San Martin de Porres, 4, 28035-Madrid, Spain
p73 is a novel member of the p53 family of tumor suppressor proteins which is involved in cellular dierentiation, tumor suppression, and the response to genotoxic stress. The molecular mechanisms regulating p73 activity are still poorly understood. Recently, p73 was found to be a target of the enzymatic activity of c-Abl, a non-receptor tyrosine kinase that potently activated in response to DNA damage. Here, we present evidence that c-Abl induces the phosphorylation of p73 in threonine residues adjacent to prolines, and that the p38 MAP kinase pathway mediates this response. Furthermore, we found that activation of p38 is sucient to enhance the stability of p73, and that the transcriptional activation of p73 by c-Abl requires the activity of p38. These ®ndings indicate that members of the MAP kinases superfamily of signaling molecules can regulate p73, and support a role for the p38 MAP kinase in a novel biochemical pathway by which c-Abl regulates this p53-related molecule. Oncogene (2002) 21, 974 ± 979. DOI: 10.1038/sj/onc/ 1205134 Keywords: p73; c-Abl; p38; p53; DNA damage Introduction The protein product of the tumor suppressor gene p53 plays a key role in maintaining the integrity of the genome by orchestrating many of the biological responses to DNA damage, including growth arrest and cell death (Kastan et al., 1991; Vogelstein and Kinzler, 1992). Indeed, functional inactivation of p53 and the consequent genomic instability may participate in the development of more than half of all human cancers. Recently, two genes known as p63 and p73 were found to encode proteins highly related to p53, whose overlapping and distinct functions in cellular dierentiation, tumor suppression, and the response to genotoxic stress has just begun to be elucidated (Kaelin, 1999; Lohrum and Vousden, 2000). In the case of p73, its protein product has been linked to development, apoptosis and tumor
suppression (Fang et al., 1999; Kaelin, 1999; Lohrum and Vousden, 1999; Steegenga et al., 1999). Moreover, p73 has been recently reported to be involved in the cellular response to DNA-damage induced by gamma radiation and chemotherapeutic agents such as cisplatin (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999), a role that was previously attributed exclusively to p53. However, it is still unclear how the functional activity of p73 is regulated under normal conditions or upon genotoxic stress. In this regard, recent evidence suggests that p73 can be a target of c-Abl (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999), a non-receptor tyrosine kinase that is linked functionally to most, if not all, key molecules involved in coordinating the response to DNA-insults, including ATM, DNA-PK and p53 (for review see Kharbanda et al., 1998; Wang, 2000). c-Abl has also been shown to stimulate a group of Stress-Activated Protein Kinases (SAPK), such as JNK (Kharbanda et al., 1995a) and p38 MAP kinase (Pandey et al., 1996). These members of the MAPK superfamily of proline-targeted serine-threonine protein kinases have also been associated with DNA-damage induced by UV light, chemotherapeutic drugs and gamma radiation (Zanke et al., 1996; for review see Ip and Davis, 1998). In turn, these SAPKs appear to play a central role in the apoptotic response to DNA damage, a function that may involve the phosphorylation of p53 (Bulavin et al., 1999; Fuchs et al., 1998; Sanchez-Prieto et al., 2000). As JNK and p38 are downstream eectors of c-Abl, and recent evidence suggests that p38 activation is required for the ability of c-Abl to induce apoptosis (Cong and Go, 1999), we decided to explore the possibility that these SAPKs could play a role in the activation of p73 by c-Abl. In this report, we show that p73 can be regulated directly by the p38 MAP kinase pathway. Furthermore, we provide evidence that the p38 MAP kinase is required for the activation of p73 by c-Abl, and that this process involves the stabilization of p73 upon phosphorylation by p38.
Results and discussion *Correspondence: JS Gutkind; E-mail:
[email protected] 3 Both authors contributed equally to this work Received 29 May 2001; revised 19 October 2001; accepted 31 October 2001
c-Abl increases the levels of p73 when expressed in 293T To examine whether exogenous p73 is aected by the expression of c-Abl, 293T cells were transfected with HA-
Activation of p73 through the p38 R Sanchez-Prieto et al
p73 and dierent amounts of c-Abl wild type (c-Abl wt) or its activated mutant lacking the SH3 domain, c-Abl act (Mayer and Baltimore, 1994). Twenty-four hours later, p73 expression was assayed by Western blotting against HA. As these human epithelial kidney cells were immortalized with Ela, we used the endogenous levels of this oncoprotein as a loading control. As shown in Figure 1a, HA-p73 was readily detectable in cells transfected with its expression plasmid. Furthermore, the expression levels of p73 increased in a dose dependent fashion when co-expressed with wt or active c-Abl. These data are aligned with previous reports indicating that c-Abl can induce the stabilization of the p73 tumor suppressor (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999). c-Abl induces the phosphorylation of p73 on tyrosine and threonine residues Taking into account that c-Abl exhibits an intrinsic tyrosine kinase activity, we next examined if p73 is tyrosine phosphorylated in 293T because of c-Abl overexpression. As previously described (Agami et al., 1999), the co-expression of c-Abl results in the tyrosine phosphorylation of the epitope-tagged p73, as judged by the use of anti-phosphotyrosine antibodies (Figure 1b). To explore whether c-Abl induces the phosphorylation of other residues on p73, the immunoprecipitated p73 was blotted with antibodies speci®c for phosphorylated threonines (thr) and serines (ser). Although we were unable to demonstrate phosphorylation on serine, we found that c-Abl expression causes a
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detectable phosphorylation of p73 on threonine residues (Figure 1b). As c-Abl does not exhibit a protein serine-threonine kinase activity, this observation indicated that kinase(s) other than c-Abl could phosphorylate p73, in a c-Abl dependent fashion. The presence of a carboxyl proline residue is strictly required for the phosphorylation of serine and threonine residues by all members of the MAP kinase superfamily. Thus, we took advantage of the availability of a speci®c antibody against phosphorylated threonine adjacent to proline to investigate the possibility that MAP kinases may mediate p73 phosphorylation. As shown in Figure 1b, this approach revealed that p73 is detectable phosphorylated in theronine residues adjacent to proline upon overexpression of c-Abl. Similar results were obtained in the presence of a constitutively active form of c-Abl (data not shown), thus raising the possibility that proline-directed serine/threonine kinases might participate in the activation of p73 by c-Abl.
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A role for p38 MAP kinase in p73 phosphorylation Among the several candidates of proline directed kinases, the JNK and p38 MAP kinase pathways have been proposed to be activated by c-Abl (Cong and Go, 1999; Kharbanda et al., 1995b). Indeed, expression in 293T cells of a constitutively active form of c-Abl is able to stimulate both JNK and p38 (data not shown). However, expression of c-Abl wt in these cells caused a clear activation of the p38 MAP kinase pathway but not
Figure 1 (a) 293T cells were transfected with the indicated amounts of pcEFL c-Abl wild type (wt) or its activated form (act) (kindly supplied by Dr B Mayer, Children's Hospital, Boston, MA, USA) ranging from 1 to 4 mg, together with 1 mg of pcDNAIII p73 a (p73) tagged with HA (kindly supplied by Dr WG Kaelin, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA) using Lipofectamine (Gibco ± BRL). Twenty-four hours later, cells were lysed in RIPA buer and analysed by Western Blotting against HA (Babco), c-Abl (Pharmingen) and E1a (Oncogene Science). Immunoblotting against E1a was performed as a loading control. (b) 293T cells were transfected as above with c-Abl wt or a plasmid control together with HA-p73, as indicated. Twenty-four hours later, cells were collected in RIPA buer in the presence of phosphatase inhibitors. p73 was immunoprecipitated with anti-HA and immunoblotted against phospho-tyrosine (4G10, Upstate Biotech), phospho-serine (Sigma), Phospho-threonine (Zymed), phospho-threonine adjacent to proline (New England Biolabs), and anti-HA. Similar results were obtained with the active form c-Abl (not shown) Oncogene
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of JNK, as judged by the use of antibodies recognizing their dual phosphorylated active forms (Figure 2a). The cotransfection of their upstream kinases, MKK6 and MKK1 respectively, was used as a positive control. To explore whether the stimulation of p38 participates in the activation of p73 by c-Abl, we ®rst expressed a dominant negative form of an upstream kinase for p38, MKK6-KR (Chiariello et al., 2000) or treated the cells with a speci®c inhibitor of p38 MAPK activation, SKF86002 (Lee et al., 2000), for 16 h, and examined the status of phosphorylation of p73. Remarkably, we observed that both the expression of MKK6 KR and the exposure to the p38 inhibitor abolished the phosphorylation on threonine adjacent to proline induced by c-Abl, and decreased the elevated levels of p73 in c-Abl expressing cells (Figure 2b). Furthermore, as shown in Figure 2C, the expression of MKK6 KR was also able to block the elevated levels of p73 provoked by activated c-Abl, without exerting any eect on the expression of c-Abl or proteins constitutively expressed by these cells, such as Ela.
Of interest, recently available information indicates that c-Abl participates in the stabilization of p73 in response to genotoxic agents such as cisplatin (Gong et al., 1999), and that cisplatin can potently activate p38 (Sanchez-Prieto et al., 2000). Thus, we next asked whether inhibition of p38 might prevent the accumulation of p73 upon exposure of cells to this chemotherapeutic agent that activates c-Abl. Indeed, as shown in Figure 2d, we observed that the treatment with cisplatin results in increased levels of p73 and the activation of p38, and that the blockade of p38 prevented the accumulation of p73 in response of cisplatin. Together, these data provided evidence that the activation of p38 by c-Abl and by cisplatin may be necessary to increase the protein levels of p73. Activation of p38 is sufficient to induce the phosphorylation of p73 on threonine residues As inhibition of the p38 pathway prevented p73 threonine phosphorylation when elicited by c-Abl, we
Figure 2 (a) 293T cells were transfected with increasing amounts of c-Abl wt and 1 mg of HA-p38 or HA-JNK. Twenty-four hours later cells were collected in lysis buer and subjected to immunoprecipitation with anti-HA. Immunoprecipitates were immunoblotted with anti-phospho p38 or phospho JNK (New England Biolabs). Co-transfection with active upstream kinases, MKK6 and MEKK1, were used as a positive control for p38 and JNK, respectively. (b) 293T cells were transfected with 2 mg of wt c-Abl and 1 mg p73 in the presence or absence of a dominant negative MKK6 (MKK6 KR). Cells were treated with vehicle control (DMSO, 0.1%) or the p38 inhibitor SKF-86002 (Calbiochem) (30 mM) for 18 h and lysed. Anti-HA immunoprecipitates were subjected to immunoblotting with phospho-threonine adjacent to proline (New England Biolabs) and HA (Babco), as indicated. (c) 293T cells were transfected with 2 mg of plasmid control (C) or the wt and the active form of c-Abl together with 1 mg p73 in the presence or absence of the same amount of a dominant negative MKK6 (MKK6 KR). Lysates were collected and analysed as in Figure 1A. Similar results were obtained in three independent experiments. (d) 293T cells were transfected with 0.5 mg p73 using lipofectamine, and 24 h later cells were preincubated with SKF-86002 (30 mM) for 1 h prior to treatment with cisplatin (Brystol Myers) (20 and 40 mg/ml) for 12 h. Lysates were collected and analysed by Western blotting against HA, E1a, P-p38, and p38, as in Figure 1a Oncogene
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asked whether activation of p38 by expression of MKK6 wt (MKK6) could mimic the eects observed by expressing c-Abl. As shown in Figure 3a, transfection of plasmids for MKK6 was sucient to activate p38 and to induce the phosphorylation of p73 on threonine adjacent to proline in a dose dependent fashion, with a concomitant increase in the amount of p73 protein. In order to analyse the role of others SAPK family member that can be activated by genotoxic stress, we asked whether an upstream activator of JNK, such as MKK7, was also able to induce p73 phosphorylation. The speci®city of MKK6 and MKK7 for the p38 and the JNK pathways, respectively, was con®rmed under our present experimental conditions. As shown in Figure 3a, MKK7 eectively induced the phosphorylation of p73 on threonine adjacent to proline, and provoked the accumulation of p73. This fact indicated that the JNK pathway is also able to control p73. However, c-Abl wt did not induce JNK but stimulated p73, and inhibition of p38 MAP kinase prevented the phosphorylation and stabilization of p73 by c-Abl and upon cisplatin treatment. Thus, although JNK may play a role in signaling to p73, available evidence points to a more prominent role for p38 under our experimental
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conditions, which therefore became the focus of further investigation. We next asked if p73 is a direct substrate of the p38 MAP kinase. For these experiments, cells were transfected with p73 and p38, both molecules tagged with HA, in the presence of an upstream activator of p38, MKK6 (Han et al., 1996). Lysates were subjected to immunoprecipitation with anti-HA and an in vitro immunocomplex kinase reaction was performed. As shown in Figure 3b, only cells transfected with MKK6 were able to render a phosphorylated p73 when coimmunoprecipitated with p38 and exposed to the phosphotransferase activity of p38. Myelin basic protein (MBP) was also included in the reaction mix and used to monitor the enzymatic activity of p38. The speci®city of this approach was con®rmed by the use of the dominant negative MKK6, which did not induce the phosphorylation on p73 (data not shown), and SB203580 that inhibits potently p38 a (Lee et al., 2000), the transfected isoform, which prevented the phosphorylation of both p73 and MBP when added to the enzymatic reaction (Figure 3b). In turn, to ensure the speci®city of the p38 inhibitor, 293T cells were also transfected with a HA-p38 MAP kinase in which threonine 106 was
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Figure 3 (a) 293T cells were transfected with 4 mg of active MKK6 or MKK7 together with 1 mg p73 (upper panel). Samples were collected and immunoprecipitated as in Figure 1b and blotted against phospho-threonine adjacent to proline (New England Biolabs) and HA (Babco). To con®rm the signaling speci®city of MKK6 and MKK7, 293T cells were transfected by Lipofectamine in the presence of HA-p38 (middle panel) or HA-JNK (lower panel). Samples were collected and immunoprecipitated as in Figure 1b and kinase reactions were performed as described, using as a substrate GST-ATF2 for HA-p38 or GST-c-Jun for JNK (Chiariello et al., 2000). (b) 293T cells were transfected with 1 mg of HA-p38 and HA-p73 in the presence or absence of 4 mg of active MKK6. Samples were collected and immunoprecipitated as in Figure 1b and kinase reactions were performed as described (Sanchez-Prieto et al., 2000), using as a substrate the co-immunoprecipitated p73 or exogenously added MBP in the absence or presence of SB203580 (5 mM). A mutant form of p38, p38 T106M (right panel), which is insensitive to this p38 inhibitor was used in parallel as a control. Phosphorylated p73 and MBP are indicated. Total immunoprecipitated HA-p38 was revealed by anti-HA Western blots. (c) 293T cells were transfected with 0.25 mg of p73 in the presence of GFP or MKK6 (1 mg) in six well plates. Cells were labeled and processed according to previously described procedures (Gong et al., 1999), except that the gels were transferred to nitrocellulose and exposed (upper panel) and immunoblotted against HA (lower panel) Oncogene
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mutated to methionine (T106M) thereby creating a p38 MAP kinase insensitive to SB-203580 (Gum et al., 1998; Sanchez-Prieto et al., 2000). As shown in Figure 3b, phospholabeling of p73 by this mutant p38 was not sensitive to SB-203580, together indicating that p38 can phosphorylate p73 in vitro, and that this phosphorylation requires an active p38 MAP kinase. Activation of p38 enhances the stability of p73 As activation of p38 by MKK6 is sucient to increase p73 protein levels, we examined whether this eect was related to the stabilization of the protein, using a pulsechase approach. Cells were metabolically labeled with 35 S-methionine/cysteine for 1 h, and the culture medium replaced with fresh medium containing excess of unlabeled aminoacids (methionine and cysteine, 1 mg/ml). Cells were lysed at dierent times, HA-p73 immunoprecipitated and transferred to PDF membranes and exposed to sensitive ®lms. After exposure, the same membranes were immunoblotted with antiHA antibody. As shown in Figure 3c, radiolabeled p73 is almost undetectable in control cells after 4 h, with a half-life of around 2 h. In contrast, cells expressing MKK6 showed a much higher stability in p73, which exhibited a half-life greater than 6 h. These data indicate that MKK6 and the subsequent activation of p38 are sucient to enhance the stability of p73 protein, and support a role for the p38 MAP kinase pathway in the stabilization of p73 by c-Abl. A dominant negative MKK6 blocks the activation of p73 induced by c-Abl and expression of MKK6 is able to enhance the transcriptional activity of p73 To analyse the functional consequences of the previous ®ndings, we decided to study whether MKK6 wild type and its dominant negative form can aect the transcriptional activity of p73. Using Saos-2 cells that do not express p53 (Chandar et al., 1992), we observed that expression of p73 is sucient to provoke the expression of a p21WAF1 promoter reporter system (Figure 4a). Cotransfection with wt c-Abl consistently enhanced this response by twofold. The presence of a dominant negative MKK6 was able to block the p73 transcriptional activity induced by c-Abl, and to restore it to the basal level of p73 activity. To study if the activation of the p38 MAP kinase pathway is able alone to stimulate p73-dependent transcription, cells were cotransfected with MKK6 using amounts of p73 that induce a sub maximal level of luciferase expression. As shown in Figure 4b, MKK6 provoked a clear activation of p73, around twofold, which was similar to that induced by c-Abl. These results reinforce the idea that the p38 MAP kinase may participate in the activation of the p73 tumor suppressor protein by c-Abl. Regulation of p73 by c-Abl through p38 Although p73 has been implicated in a variety of cellular processes, such as in apoptosis and tumor
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Figure 4 (a) Saos 2 cells were transfected in six well plates with the indicated DNAs (100 ng each) using Lipofectamine Plus in the presence or absence of HA-p73 (50 ng), together with a p21WAF promoter linked to luciferase (kindly supplied by Dr B Vogelstein, John Hopkins University, MD, USA) and pNullrenilla (Promega) as an internal control. Eighteen hours after transfection, cells were lysed using reporter lysis buer (Promega) and assayed for luciferase and renilla activity in a Dynex Luminometer. Data are expressed as luciferase activity normalized by the activity of renilla in each sample. Data represent the mean+s.e.m. of triplicate samples from a representative experiment that was repeated three independent times. (b) Saos-2 cells were transfected with HA-p73 expression plasmid (25 and 50 ng) in the presence of increasing amounts of MKK6 (0, 50, and 100 ng) and the luciferase activity was measured as above. Data represent the mean+s.e.m. of triplicate samples from a representative experiment that was repeated four independent times
suppression (Irwin et al., 2000; Kaghad et al., 1997; Lissy et al., 2000; Pozniak et al., 2000), the molecular mechanisms that control the activity of this protein are still poorly understood. Recent studies indicate that cAbl can promote the stabilization of p73, thereby enhancing its transcriptional activity. The ability of cAbl to promote the tyrosine phosphorylation of p73 suggested that c-Abl can act on p73 directly (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999). However, as shown above, we found that c-Abl and its activated form can also provoke the phosphorylation of p73 on threonine residues, and more particular on threonine residues adjacent to prolines, thus indicating that p73 is a candidate substrate for proline-targeted serine-threonine kinases.
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In this regard, c-Abl can promote the activation of several members of the MAPK superfamily, including MAPK, JNK, and p38. Indeed, all three kinases were potently stimulated by the activated form of c-Abl (Figure 2 and data not shown), but overexpression of cAbl, which is itself sucient to activate p73, resulted in the activation of p38 but not of JNK or MAPK under our experimental conditions. Using a number of experimental approaches, we obtained evidence that p38 can itself phosphorylate p73 in vivo and in vitro, and that activation of the p38 pathway is sucient to enhance the protein stability of p73. Furthermore, we provided evidence that the p38 MAP kinase can mediate the phosphorylation on threonine adjacent to prolines when provoked by c-Abl, and that p38 is required to stimulate the transcriptional activity of p73 by c-Abl. Accumulated evidence suggests that the JNK pathway can also promote p73 phosphorylation and stabilization. Although p38 appears to play a primary role in p73 activation in response to c-Abl and cisplatin, this observation raises the possibility that JNK may participate in signaling to p73 when elicited by other stress inducing agents and genotoxic drugs, many of which are known to activate JNK potently. Taken together, we can conclude that these results connect
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stress-activated pathway to the regulation of p73, and, in particular, support a role for p38 in a novel biochemical pathway by which c-Abl regulates this p53-related molecule. These ®ndings are expected to have broad implication in cancer treatment, as c-Abl is activated upon exposure to chemotherapeutic agents and ionizing radiation. In particular, the activation of p73 by c-Abl through p38 may play an important role in the initiation of apoptotic programs in response to cancer therapy in many tumors that lack a functional p53 but express p73 (Cross et al., 2000), a possibility that is under current investigation. This study also supports an important role for the p38 MAP Kinase pathway in cancer therapy, as previously published reports and our present results indicate that p38 can control the transcriptional activity of both p53 and p73 members of the p53 family of tumor suppressor proteins (Bulavin et al., 1999; Sanchez-Prieto et al., 2000).
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Acknowledgments We appreciate the comments of K Sakabe, C Murga, H Miyazaki, A Senderowicz, S Montaner, B Fletcher, V Patel and S Ramon y Cajal. We also appreciate the assistance and advice of L Vitale-Cross, C Parada and J Guinea.
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