Activation of p53 protein

Details Activation of p53 protein Activation of p53 protein

1995-07-24

2000-10-31

Eyler, Yvonne

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 723, 4241551, 4241741, 530350, 530358, G01N 3353, G01N 33574, A61K 39395, C07K 100

Patent

active

061400580

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to the p53 tumour suppressor protein, and more particularly to mutant forms of the p53 protein which seem to be associated with certain types of tumour, and are usually found at elevated levels in such tumour cells. The invention also discloses the conversion of wild type p53 to a constitutively active form.


BACKGROUND TO THE INVENTION

Mutation of p53 is a very common genetic alteration in human cancers (1). Loss of the p53 tumour suppressor activity is co-incident with a loss of G1-S checkpoints following DNA damage (2), increases in genomic instability and selectable gene amplification (3,4). Mutant forms of p53 also appear to acquire a dominant growth promoting function (5,6). The region responsible for this transforming activity has been localized to a small C-terminal domain (7). Mice null for the wild type p53 locus develop normally, yet are susceptible to the development of neoplasia at elevated frequencies (8), indicating that wild type p53 is dispensable for the control over normal development and cell differentiation, but is essential to prevent spontaneous tumour formation. Consistent with this data, inherited germline point mutations in p53 lead to a predisposition to cancer in humans (9).
Wild type p53 protein levels rise dramatically in response to the DNA-damaging agents mitomycin C (56) UV light (10,11) and .gamma. irradiation (2). Biochemical characterization of wild type protein has shown that p53 can function as a sequence-specific DNA binding protein (12,13) and a transcription factor (14-16). These results support a model for wild type p53 in which its function is activated post-translationally after DNA damage to allow DNA repair by controlling the expression of regulatory gene products (18). These may include the DNA damage inducible gene gadd45 (19) and the host protein with oncogenic properties mdm-2 (20).
Biochemical characterization of p53 has become possible recently owing to the use of protein expression systems which allow for an abundant source of the protein. p53 purified using immunoaffinity chromatography has been shown to be a sequence-specific DNA binding protein which recognizes a motif containing two contiguous monomers of the sequence (Pu).sub.3- C(A/T)(A/T)G(Py).sub.3 (SEQ ID NO:8) (21). Sequence-specific DNA binding activity is manifested in the ability of p53 to bind the SV40 origin of replication (13), and by its ability to activate transcription in vitro from templates harbouring its DNA binding sequence (22). Mutant forms of p53 are defective in non-specific DNA binding (23), sequence-specific DNA binding (21) and transcriptional activation (14) suggesting that this activity is normally required to suppress tumour formation.
Phosphorylation of nuclear DNA binding proteins is an effective mechanism through which gene expression is controlled in response to environmental cues (24). Multi-site phosphorylation of p53 by protein kinases (25-27) suggests its tumour suppressor activity may be tightly co-ordinated by complex signal transducing pathways.
Casein kinase II (CasKII) is a highly conserved calcium and nucleotide independent enzyme which phosphorylates a broad spectrum of substrates, including transcription factors and DNA binding proteins (28). The activity of the kinase is stimulated in cells exposed to a variety of mutagens and growth factors (29). Mouse (25) and human p53 (17) are phosphorylated at the penultimate C-terminal amino acid by Cas KII in vitro. Mutation of this highly conserved C-terminal serine residue of mouse p53 to an alanine abolishes the growth suppressive function of the protein in mammalian cells, suggesting that phosphorylation at this site is one important modification required to activate the tumour suppressor function of p53 (30). Based on biochemical and physiological data, CasKII is the only known enzyme involved in a direct and positive regulation of the activity of p53.
Understanding the regulation of p53 activity is a vital step for the development of therapeutic strategies desi

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