This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. p63, a p53 homologue, is transcribed into six isoforms derived from differential promoter usage and alternative splicing. The transactivation (TA) isoforms, which resemble p53, are generated by the use of an upstream promoter and consist of an acidic N-terminal transactivation domain, a central DNA binding domain and a C-terminal oligomerization domain. The ?N isoforms, produced from an intronic promoter, are capable of forming protein complexes with p53 family proteins to inhibit the function of TAp63 and p53 family members by sequestering or competing for target promoters it. Furthermore, the TAp63 and ?Np63 isoforms undergo alternative splicing yielding different C-termianl tails. Unlike p53, which is dispensable for normal development, p63 is critical for the development of stratified epithelial tissues, such as epidermis, breast and prostate. p63 -/- mice completely lack stratified squamous epithelial and their derivatives, including epidermal appendages and mammary lacrimal, and salivary glands. Due to the lack of an epidermal barrier, these mice dehydrate and die shortly after birth. In addition, p63 -/- mice have major defects in limb and craniofacial development. Limb truncation in p63-/- mice are due to failure to maintain or differentiate the apical ectodermal ridge, a structure required for limb extension and dependent on coordinated epithelial-mesenchymal interactions. Several autosomal dominant inherited human syndromes have have been mapped to the p63 gene. These syndromes have various combinations of limb malformations fitting the split hand-split foot spectrum, orofacial clefting, and extodermal dusplasia. p63 may also play a role in tumorigenesis. To explore these roles, we have generated GST-fusion constructs using pGEX-2TK vector through PCRbased cloning approach. We transformed these plasmids into BL21 E.Coli strain, induced by IPTG and examined the expression levels of these fusion proteins. We are purifying the GST-p63 fusion proteins using a GST-pull down approach. The purified GST-p63 fusion proteins are being used to pull down proteins from lysates of HeLa cells. Proteins are separated on SDS-PAGE and stained . Individual protein bands are destained, protease digested and subjected to MALDI-MS and LTQ-Orbitrap ESI-MS/MS analysis.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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