Understanding the genetic basis of mammalian development is not only important from a basic biological view but is also relevant to human disease. The development of the mammalian embryo from a single fertilized egg utilizes the entire spectrum of genetic and biochemical regulatory mechanisms. Pluripotent precursor cells, or stem cells, must proliferate to renew the embryonic population and also differentiate to generate the highly specialized cell types unique to particular tissues and structures. Many types of human diseases result from the aberrant proliferation of cells that appear more de-differentiated, assuming an embryonic phenotype. In cancer, such dedifferentiated cells are also prone to migration and invasion, two processes oft seen in the developing embryo. Growth and differentiation of precursor cells are controlled both by intrinsic proteins, such as transcriptional activators and repressors, and by extrinsic factors, such as secreted cell signaling molecules. The interplay between cell-cell signaling and gene activation by transcription lies at the heart of genetic regulatory mechanisms controlling differentiation, proliferation, cell death, and morphogenesis. The developing kidney is an excellent model system to study epithelial cell differentiation and morphogenesis of a complex organ system. Pax2 is a transcription factor transiently expressed in the early kidney precursor cells, the metanephric mesenchyme, and in the proliferating epithelial derivatives of this mesenchyme. While Pax2 is absolutely essential for kidney development, failure to suppress Pax2 expression in more differentiated renal tubules is associated with a variety of disease including renal cell carcinoma, polycystic kidney disease, and juvenile cystic dysplastic kidneys. The activity of Pax2 is stimulated by phosphorylation of the transactivation domain by the c-Jun N-terminal kinase (JNK). Furthermore, the interaction of Pax2 with the Groucho family of transcriptional repressor molecules inhibits phosphoprylation of the Pax2 activation domain. Thus, Pax2 activity is regulated by both extrinsic signaling, through JNK, and by intrinsic nuclear factors, such as Groucho, to control the activation or repression of downstream Pax2 target genes. This proposal will map the specific serine residues of Pax2 phosphorylation within the large activation domain. Phospho-Pax2 specific antibodies will be generated to localize the active form of the Pax2 protein in vivo and to determine the interactions of Phospho-Pax2 with the cellular transcription machinery. We will also address the role of the Pax2 interacting protein PTIP in modulating Pax2 activity. PTIP is an essential nuclear factor for cell proliferation that associates with actively expressed chromatin. Given the role of Pax2 in the proliferation of renal epithelial cells and in renal disease, controlling Pax2 activity through its interactions with other cellular proteins can potentially lead to novel therapeutic interventions for cancer, PKD and other kidney disease. ? ?
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