This is a new application by an established investigator, who is searching for Pax-2 interacting proteins, in order to more fully understand Pax-2 function. Pax-2 is a paired domain transcription factor, critical in renal development, where it is induced in metanephric mesenchyme by ureteric bud stimuli, and functions in mesenchymal to epithelial cell transition. Pax-2 is repressed by WT-1 in differentiated renal tubular epithelium, but is re-expressed in Wilm's tumor, renal cell Ca, and PKD. The preliminary data shows that a novel protein termed PTIP (pax transactivation domain interacting protein) - which contains five BRCT domains - interacts with Pax-2 in a domain-specific fashion as defined by a). the yeast two hybrid system, b). pull-down experiments with Pax-2 GST fusion proteins and c). by co-immunoprecipitation after transient co-transfection in NIH 3T3 cells using various deletion mutants of Pax-2. The human PTIP homolog has been defined, the gene assigned to chromosome 7, and the extensive distribution in tissues was shown.
Aim 1 of this proposal is to define the domains in PTIP that interact with Pax-2. This will be done using the yeast-2 hybrid approach and pull-down experiments with deletion mutant-GST fusion proteins. Once domains are defined, point mutations will be made to further define the interaction. Based on information obtained, dominant negative mutants will then be made, which should be useful in defining the function of endogenous PTIP. The dominant negative mutants will be used to determine whether they inhibit Pax-2 promoter-reporter activation. Overexpression of PTIP has already been attempted, and did not alter Pax-2 promoter activation. It is hypothesized that this may be due to the already abundant expression of endogenous PTIP.
Aim 2 is based on the preliminary data showing co-immunoprecipitation of four prominent proteins with antibodies directed against PTIP. It is proposed that these be purified, sequenced, and thus identified. Purification from rat brain, which expresses abundant PTIP will be done using an immunoaffinity approach with PTIP antibodies. PTIP interacting proteins will also be sought by co-immunoprecipitation of nuclear fractions with myc-tagged over expressed PTIP in 3T3 cells. In addition to co-immunpurification, the yeast-2 hybrid approach will be used to look for PTIP interacting proteins.
Specific Aim 3 is based on the preliminary finding that PIAS-3, an inhibitor of Stat3 DNA binding and transcriptional activation, interacts with Pax-2 in the yeast 2 hybrid system. Hence, it will first be determined whether PIAS-3 has similar inhibitory activity on Pax-2 promoter reporter expression. Given the known action of PIAS-3 to inhibit Stat-3 DNA binding, the possibility that PIAS-3 also inhibits the Pax-2 - DNA interaction will be examined using EMSA. If PIAS3 indeed inhibits Pax-2 transcriptional activity and/or DNA interaction, then an interaction with other Pax family members will be sought. Another component of this aim is to seek an interaction of Pax-2/PIAS-3 with Stat-3 dependent transcriptional activation. As EGF induces Stat-3 dependent gene transcription, it is proposed that Pax-2 could act by competitively binding PIAS-2, thus relieving the inhibition on Stat-3. This component of Aim 3 will be done in MDCK cells, in which the effect of over expressed PIAS-3, Pax-2, alone or in combination, on EGF- and HGF stimulated Stat-3 phosphorylation and DNA binding will be determined. Stat-3 activation will be examined by immunoprecipitation of overexpressed myc-tagged Stat-3, followed by blotting with antiphosphotyrosine. Stat-3 dependent activation of an appropriate promoter-reporter construct will also be sought.
Specific Aim 4 it is proposed to seek additional Pax-2 interacting proteins, using an embryonic kidney library expressed in the yeast-2- hybrid prey vector. In particular, proteins interacting with the Pax-2 octapeptide, so important in reducing Pax-2 activity, will be sought. Interactions will be confirmed using GST-pulldown experiments, and Pax-2 HA co-immunoprecipitation. Interaction with Pax-6 and Pax-8 will be sought in the yeast 2 hybrid system. Expression patterns will be examined using Northern blots and in situ hybridization, with particular emphasis in embryonic kidney. Antibodies will be made, and interaction domains with Pax-2 will be defined as has already been done with PTIP.
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