Margaret Ashcroft

Regulation of p53 Function and Stability by Phosphorylation

ABSTRACT The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

Regulation of p53 stability

Leading the way in imposing a policy of zero tolerance of cellular abnormalities that might lead to tumor development is the p53 protein. The efficiency of p53 in preventing cell growth is a strong deterrent to malignant progression, but this activity must be kept tightly restrained to allow normal cell growth and development. Essential components of this regulation are the mechanisms by which the p53 protein is degraded, and efficient turnover of p53 in normal cells prevents the accumulation of the protein. Modulation of these degradation pathways in response to stress leads to the rapid stabilization and accumulation of p53, and activation of the p53 response. It is now becoming clear that the Mdm2 protein is central to the regulation of p53 stability and multiple pathways exist through which the activity of Mdm2 can be inhibited. Defects in the ability to stabilize p53 are likely to contribute to malignant development, and restoration of this activity represents an extremely attractive possibility for tumor therapy.

Stress Signals Utilize Multiple Pathways To Stabilize p53

ABSTRACT The p53 tumor suppressor is activated by many diverse stress signals through mechanisms that result in stabilization and accumulation of the p53 protein. p53 is normally degraded through the proteasome following interaction with MDM2, which both functions as a ubiquitin ligase for p53 and shuttles to the cytoplasm, where p53 degradation occurs. Stabilization of p53 in response to stress is associated with inhibition of MDM2-mediated degradation, which has been associated with phosphorylation of p53 in response to DNA damage or activation of ARF. In this study we show distinct responses, as measured by phosphorylation, transcriptional activity, and subcellular localization, of p53 stabilized by different activating signals. Although normal cells and wild-type p53-expressing tumor cells showed similar responses to actinomycin D and camptothecin treatment, the transcriptional activity of stabilized p53 induced by deferoxamine mesylate, which mimics hypoxia, in normal cells was lost in all three tumor cell lines tested. Our results show that multiple pathways exist to stabilize p53 in response to different forms of stress, and they may involve down-regulation of MDM2 expression or regulation of the subcellular localization of p53 or MDM2. Loss of any one of these pathways may predispose cells to malignant transformation, although reactivation of p53 might be achieved through alternative pathways that remain functional in these tumor cells.

A ribonucleotide reductase gene is a transcriptional target of p53 and p73

Many p53-inducible genes have been identified that might play a role in mediating the various downstream activities of p53. We have identified a close relative of ribonucleotide reductase, recently named p53R2, as a p53-inducible gene, and show that this gene is activated by several stress signals that activate a p53 response, including DNA damaging agents and p14ARF. p53R2 expression was induced by p53 mutants that are defective for the activation of apoptosis, but retain cell cycle arrest function, although no induction of p53R2 was seen in response to p21WAF1/CIP1-mediated cell cycle arrest. Several isoforms of the p53 family member p73 were also shown to induce p53R2 expression. Transient ectopic expression of either wild type p53R2 or p53R2 targeted to the nucleus, did not significantly alter cell cycle progression in unstressed cells. The identification of this gene as a p53 target supports a direct role for p53 in DNA repair, in addition to inhibition of growth of damaged cells.

Regulation of Mdm2-directed degradation by the C terminus of p53.

ABSTRACT The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.

Identification of a cryptic nucleolar-localization signal in MDM2

he tumour suppressor protein p53 is a potent inhibitor of cell growth, activating both cell-cycle arrest and apoptotic pathways in stressed cells. One of the principal regulators of p53 function and stability is the MDM2 protein, which can target p53 for ubiquitin-dependent degradation by the proteasome, as well as regulating its own stability. p53 is stabilized in response to stress through several mechanisms, including the expression of ARF (p14 in humans and p19 in mouse) in response to abnormal proliferative signals. The ARF protein binds directly to MDM2 and blocks p53 degradation by inhibiting the E3 ubiquitin-ligase activity associated with MDM2 (ref. 8) and preventing nuclear export of MDM2 (refs 9, 10). The ability of ARF to inhibit MDM2 function is related to the localization of ARF to the nucleolus, and nucleolar-localization signals (NoLSs) have been identified in ARF. Here we identify an NoLS in the carboxy-terminal region of human MDM2 that does not function in unstressed cells, but is necessary to cooperate with nucleolar-localization signals in p14 to allow relocalization of both proteins. We studied the localization of a series of human MDM2 mutants in human cell lines that do not express p14 (ref. 6), namely U2OS cells (Fig. 1a) and MCF-7 cells (data not shown). As seen previously, in the absence of ARF, full-length MDM2 was expressed predominantly in the nucleoplasm of the transfected cells, with evidence of nucleolar exclusion. An MDM2 mutant lacking the C-terminal RING-finger domain (amino acids 1–440) and an MDM2 protein carrying a point mutation in the RING finger (at Ala 464) were also localized in the nucleoplasm, like full-length MDM2. Both of these RING-finger mutants are defective in ubiquitin-ligase activity and fail to degrade p53 (refs 8, 12). These two mutants are also stable themselves, reflecting a role for MDM2 in regulating its own stability, and are expressed at equivalent levels following transfection (data not shown). An MDM2 mutant that is deleted of the central region of the protein (MDM2 ∆222–437) showed nucleolar localization, although low levels of nucleoplasmic expression could be seen in most cells. Finally, expression of an MDM2 mutant deleted of the nuclear-import signal (∆150–230) showed cytoplasmic localization, as previously described. The nucleolar expression of MDM2 ∆222–437 in these ARF-null cells indicated that MDM2 may itself contain an NoLS, and examination of the sequences retained in the MDM2 ∆222–437 mutant revealed the presence of a stretch of basic residues within the C-terminal region that might function in this way (Fig. 1b). To test the ability of these sequences to function in nucleolar localization, we expressed a peptide corresponding to MDM2 residues 466–473 in the active-site loop of thioredoxin. The resulting protein contained nuclear-localization signals derived from SV40 and a Mycepitope tag, to facilitate detection. The thioredoxin protein expressed alone was localized to the nucleus and showed general nucleoplasmic staining, with nucleolar exclusion (Fig. 1c). However, introduction of MDM2 residues 466–473 resulted in the reloT

Phosphorylation of HDM2 by Akt

The HDM2 protein is a key regulator of the tumour suppressor, p53. Control of HDM2 function is critical for normal cell proliferation and stress responses, and it is becoming evident that multiple modifications of HDM2 can regulate its function within cells. In this study we show that HDM2 associated with the serine-threonine kinase, Akt, in response to growth factor stimulation of human primary cells. This association was concurrent with phosphorylation of Akt (at Ser 473), and resulted in elevated expression of HDM2 and enhanced nuclear localization. However, analysis of HDM2 proteins mutated at the consensus Akt recognition sites at serines 166 and 186 indicated that modification at these residues was not sufficient for the increased expression of the protein, which was blocked by the PI3 kinase inhibitor LY294002. Tryptic peptide and mutational analyses revealed evidence for an Akt phosphorylation site in HDM2 additional to the two consensus sites.

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trkdef) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-γl, Ras, PI 3-kinase and SNT. Trkdef was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trkdef. NGF-stimulated tyrosine phosphorylation of the Trkdef+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trkdef+PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trkdef+PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Contribution of two independent MDM2-binding domains in p14ARF to p53 stabilization

The MDM2 protein targets the p53 tumor suppressor for ubiquitin-dependent degradation [1], and can function both as an E3 ubiquitin ligase [2] and as a regulator of the subcellular localization of p53 [3]. Oncogene activation stabilizes p53 through expression of the ARF protein (p14(ARF) in humans, p19(ARF) in the mouse) [4], and loss of ARF allows tumor development without loss of wild-type p53 [5] [6]. ARF binds directly to MDM2, and prevents MDM2 from targeting p53 for degradation [6] [7] [8] [9] by inhibiting the E3 ligase activity of MDM2 [2] and preventing nuclear export of MDM2 and p53 [10] [11]. Interaction between ARF and MDM2 results in the localization of both proteins to the nucleolus [12] [13] [14] through nucleolar localization signals (NoLS) in ARF and MDM2 [11] [12] [13] [14]. Here, we report a new NoLS within the highly conserved amino-terminal 22 amino acids of p14(ARF), a region that we found could interact with MDM2, relocalize MDM2 to the nucleolus and inhibit the ability of MDM2 to degrade p53. In contrast, the carboxy-terminal fragment of p14(ARF), which contains the previously described NoLS [11], did not drive nucleolar localization of MDM2, although this region could bind MDM2 and weakly inhibit its ability to degrade p53. Our results support the importance of nucleolar sequestration for the efficient inactivation of MDM2. The inhibition of MDM2 by a small peptide from the amino terminus of p14(ARF) might be exploited to restore p53 function in tumors.

Nerve growth factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoinositide 3-OH kinase

The TrkA receptor protein tyrosine kinase is involved in signalling PC12 cell differentiation and cessation of cell division in response to nerve growth factor (NGF). To assess the importance of adaptor proteins and Ras in NGF control of phosphoinositide 3-OH kinase (PI 3-kinase), specific receptor mutations in Trk have been employed. We show that phosphorylation of tyrosine 490, but not 785, of Trk is essential for activation of both Ras and PI 3-kinase in vivo, correlating with tyrosine phosphorylation of Shc and binding of Shc to the adaptor Grb2 and the Ras exchange factor Sos. A mutant receptor that lacks Y490 and Y785, but contains an introduced YxxM motif which binds the regulatory domain of PI 3-kinase, is unable to activate Ras despite causing increased PI 3-kinase activity. This indicates clearly that activation of PI 3-kinase by itself is not sufficient to cause activation of Ras, arguing against a model in which PI 3-kinase acts upstream of Ras. The Shc site of Trk is thus crucial for the activation of Ras and PI 3-kinase.