The mdm2 oncogene encodes a 90 kDa protein that binds to the p53 tumor suppressor protein and negatively regulates its function in transcription, cell cycle arrest and programmed cell death (apoptosis). MDM2 gene amplification and overexpression occur in 40-50% of osteosarcomas and 30% of soft tissue sarcomas in humans. We have found that cells undergoing p53-mediated apoptosis activate a protease that cleaves the MDM2 protein into two fragments. The cleavage site on MDM2 has been identified and indicates the involvement of an ICE-like protease (Interleukin 1-converting enzyme). The cleavage by the protease and the cleavage site sequences are conserved in evolution, suggesting it is an important feature of MDM2. They hypothesize that the cleavage of MDM2 by an ICE family protease during apoptosis may be a critical metabolic step with the following outcomes: (a) To inactivate its p53 binding function by altering the structure of the protein. (b) Inactivate functions of MDM2 that normally promotes cell survival. (c) Activate new functions of MDM2 that participate in the induction of cell death. To test these hypotheses, the aims of the proposed experiments are: (1) Identify the apoptotic protease that cleaves MDM2 and study its activation mechanism during apoptosis. (2) Determine the biochemical properties of the MDM2 cleavage fragments. (3) Examine the ability of the cleaved MDM2 to regulate p53 functions. (4) Determine the functional significance of MDM2 cleavage in apoptosis. The ICE family proteases play critical roles during apoptosis, probably by activation or inactivation of multiple substrates. MDM2 is the first oncogene product in the p53 pathway found to be the target of these proteases. Investigating this phenomenon in detail will provide a better understanding of the mechanism of the ICE family proteases and the function of MDM2 in apoptosis.
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