STRUCTURE FUNCTION RELATIONSHIP OF TUMOR SUPRESSORS

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DESCRIPTION: (Verbatim from the Applicant's Abstract) The objective of this project is to understand the structure-function relationship of newly discovered tumor suppressors, particularly those in the p53-Rb pathway. The focus of the last granting period was on p16-INK4A. There are three major goals for the next granting period. Specific Aim 1 is to extend previous studies on the specificity of the interactions between INK4 protein and cyclin-dependent kinase 4 (CDK4) or CDK6 in two directions: (a) Site-directed mutagenesis will be used to probe the structural and functional roles of a number of surface residues that have contrasting charges among different INK4 proteins. (b) Gene shuffling will continue to be used to obtain new INK4 variants with altered specificity, and the properties of new constructs with most interesting properties will be characterized at the protein level. As part of these studies, two additional assay methods will be developed to complement the inhibitory assay currently in use: a fluorescent binding assay for quantitative determination of INK4-CDK dissociation constants, and an in vivo assay for INK4 proteins in cancer cell lines. Specific Aim 2 is to extend the studies of INK4 proteins to their interactions with human HTLV-1 Tax protein. The applicant plans to determine the structures of Tax delta109 and use site-directed mutagenesis to identify key residues from both Tax and p16 that are involved in the interactions, and compare p16-Tax interactions to p16-CDK4 (or CDK6) interactions. Specific Aim 3 is to extend the studies to other new proteins relevant to the p53-Rb pathway. The two proteins to be pursued are human p14ARF, a tumor suppressor expressed from the same gene locus as p16 but in an alternate reading frame, and human TRIP-Br1, a member of a newly discovered novel family of transcription factors that has been found to be involved in the transcriptional machinery of E2F-1. The approach involves a combination of genetic, biochemical and biophysical techniques.