The Hedgehog (Hh) pathway is an evolutionarily conserved signaling pathway that plays fundamental roles during embryogenesis and in adult stem cells. Dysregulation of Hh signaling has severe consequences in humans, ranging from birth defects to the development of basal cell carcinoma, medulloblastoma, and many other tumors. Many human disease-associated mutations have been identified in Hh pathway components. Regulation of the Hh pathway has attracted considerable attention from both basic and clinical-scientists. The Hh pathway can be regulated at multiple levels. The Gli family of transcription factors act at the end of the Hh signaling cascade to control the expression of Hh target genes. Therefore, it is possible that abnormalities in the Hh pathway can be corrected by modulating the function of Gli proteins in human patients. However, although many regulators of the Hh pathway have been identified, it remains largely unclear how the transcriptional activation function of Gli is regulated. Genetic studies in zebrafish have identified Daz interacting protein 1 (Dzip1) as a regulator of Gli. Based on phenotypic characterization of zebrafish mutants deficient in Dzip1, it was proposed that Dzip1 regulates the function of Gli by inhibiting Suppressor of Fused (SuFu), an inhibitor of Gli. However, this hypothesis has not been tested molecularly. Recent studies in Drosophila and our work in vertebrates have demonstrated that B56e, a regulatory subunit of protein phosphatase 2A (PP2A), regulates the transactivation function of Gli through an as yet unclear mechanism. In an effort to understand how B56e regulates Gli activity, we found that B56e and Dzip1 physically interact. We were able to show that Dzip1 synergizes with Gli1 in mammalian culture cells and is required for the expression of Hh target genes in Xenopus embryo. Most strikingly, we found that Dzip1 forms a complex with SuFu in mammalian culture cells. These preliminary results raise the intriguing possibility that Dzip1 and B56e, which directly interact with each other, are involved in an important Gli regulatory pathway. Interestingly, inhibition of protein kinase A (PKA) in zebrafish promotes nuclear localization of Dzip1, indicating that Dzip1 is regulated by the antagonistic interaction between protein kinase(s) and protein phosphatase(s). Thus, we hypothesize that B56e regulates Gli activity through dephosphorylating Dzip1.
Specific aims are:
Aim1 : To determine whether B56e regulates Dzip1 phosphorylation.
Aim2 : To determine whether B56e regulates the function of Dzip1 in the Hh pathway.
Aim3 : To determine whether B56e antagonizes the function of SuFu.
Mutations in human Hh pathway components cause birth defects and tumorigenesis. In principle, abnormalities in the Hh pathway can be corrected by modulating the activity of Gli proteins in human patients. Our preliminary studies have led to the observation that Dzip1 and B56e, which regulate Gli activity, directly interact with each other. We design experiments here to test the hypothesis that Dzip1 and B56e are involved in a novel Gli regulatory pathway. These studies will likely uncover an important mechanism of Gli regulation and accelerate the development of tools for correcting Hh signaling in human patients.
