Ute Moll, Ph.D. (Professor)

The p53 gene plays a pivotal role in DNA damage signaling pathways in mammalian cells. Upon receiving a damage signal, p53 protein undergoes nuclear accumulation and posttranslational modification to become functionally activated. Research in my laboratory has focused on 2 related issues i the regulation of p53 activity in normal cells and ii) its dysregulation in certain cancer cells.

A) P53 regulation in normal cells. We investigated whether p53'srequirement for nuclear access constitutes an important level of regulation of its activity as a transcription factor. The existence of such a spatialmechanism of p53 regulation was suggested by 2 facts:
1) p53 in normalmouse ES cells is sequestered in the cytoplasm and functionally inactive and

2) subcellular localization of p53 in normal unstressed cells underlies cell cycle regulation. P53 is nuclear in G1 and cytoplasmic during S and G2, consistent with its role as a mediator of G1 checkpoints. Conversely, an effective means of down regulating p53 activity is through its spatial separation from its downstream effector genes. In collaboration with G. Wahl (Salk Institute), we identified a novel NES (nuclear export signal) within the tetramerization domain of p53. The p53 NES consists of aleucine-rich sequence which conforms to NES criteria and which is highly conserved among widely divergent species and among the p53 homologs p63 andp73. This intrinsic NES mediates subcellular localization and nuclear-cytoplasmic shuttling of p53 through association with an exportreceptor, most likely CRM1. Mutation analysis of the p53 NES abolished the export ability concurrently with efficient tetramer formation. Although the p53 antagonist MDM2, which binds to p53 and mediates its degradation atcytoplasmic proteasomes, has its own NES and has been proposed to mediatep53 nuclear export, we showed that MDM2 is not required for p53 to exit the nucleus. We propose a model in which subcellular localization of p53is established through tetramerization-regulated exposure of the p53 NES to the export machinery. Nuclear retention is regulated by its conversion from DNA-binding tetramers with buried (masked) NES to non-binding monomers ordimers with exposed NES. This model provides a novel form of regulation ofp53 activity and provides a mechanism for simultaneous nuclear retention and tetramerization of p53, both of which are required for p53 function. It also provides a new direction in the search for cellular activities thatare important in down regulating a p53 stress response after the damage hasbeen repaired.

B) P53 dysregulation in cancer. A long-term interest of my lab, that ultimately led to the work described in section A, lies with our discovery of a mutation-independent mechanism of p53 inactivation in certain cancers. Generally, inactivating lesions in the p53 pathway that are critical for tumorigenesis fall into 3 classes. The most frequent class consists of intragenic mutations that perturb the structure of the sequence-specific DNA binding domain of p53, thereby incapacitating its transactivationability of target genes that mediate its tumor suppressor functions. A second class comprises extragenic lesions affecting proteins that regulate the activity of the p53 protein, such as MDM2 and p19ARF. We discovered a third class of inactivating lesions that result in nuclear exclusion of wild type (wt) p53 in breast cancer and neuroblastoma, an aggressive childhood neoplasm. Constitutive cytoplasmic accumulation of wt p53 strictly correlates with malignancy, since 95% of malignant neuroblastomasexhibit this abnormality but none of the biologically benign derivative tumors that had undergone differentiation. Later, others described this phenotype in colon carcinoma, retinoblastoma, other types of breast cancerand embryonic stem cells (ES). Also, in liver cancers associated with hepatitis B virus (HBV) infection, the viral protein-X directly contributes to hepatocellular transformation by sequestering wild type p53 in thecytoplasm. This results in blockage of p53 entry into the nucleus, inhibition of its transactivation activity and p53-mediated apoptosis(Feitelson et al).

We determined that cells with cytoplasmically sequestered wild type p53 are poorly responsive or non responsive to upstream signals that would normally induce its activity. Neuroblastoma cells have an impaired G1 arrest and are completely incapacitated for p53-mediated apoptosis after DNA damage. Others showed that ES cells do not activate p53-dependent stress responses after DNA damage. Recently we identified the mechanism underlying the nuclear exclusion and cytoplasmic sequestration of wild type p53 in neuroblastoma cells. Despite the appearance of static cytoplasmic sequestration, we showed that this is not due to the action of a cytoplasmic tether mechanism or a defect in nuclear import. Rather, p53 in these cells is subject to continuous nuclear-cytoplasmic shuttling but with predominant nuclear export due to a dysregulation of the NES/tetramerization switch. This hyperactive export could be overcome by masking the intrinsic NES of p53 via forced tetramerization of endogenous p53. Nuclear retention of p53 in these cells was achieved by co-expressing C-terminal peptides containing the tetramerization domain but not with tetramerization mutants.

Other research interests:

We are currently investigating the role of the p53 related gene family members in human cancers. Despite structural and, in ectopic expression systems, functional homolgy to p53, these genes do not appear to be Knudson type tumor suppressor genes. Their current role is currently unknown. Curiously, tumors overexpress wild type gene products in multiple isoforms (alternate splicing and promoter use).