Phosphorylation Modulates PITX2 and ARS Mutant Transcriptional Activities: A Role for Protein Kinase C (PKC) in Tooth Development?

PITX2 is the first transcriptional marker observed during tooth development and PITX2 mutations associated with Axenfeld-Rieger syndrome (ARS) provided the first link of this homeodomain transcription factor to tooth development. Objectives: PITX2 contains multiple PKC sites and its activity is regulated through phosphorylation by PKC. We are studying the role PKC phosphorylation plays in regulating PITX2 transcriptional activity. Furthermore, we have identified a new ARS mutation that produces a consensus PKC site in the homeodomain and results in tooth anomalies in this patient. Methods: we use electrophoretic mobility shift assays, mutation analysis, kinase activators and inhibitors, transient transfections, immunochemical and biochemical assays to analyze PITX2 phosphorylation and transcriptional activation. Results: PITX2A contains 10 PKC sites; three in the N terminus, two in the homeodomain and five in the C terminus. We have mutated all ten sites individually and in combinations to demonstrate the contribution of PKC phosphorylation to the transcriptional activity of PITX2. Mutation of the C-terminal PKC sites results in reduced transcriptional activity while N-terminal PKC mutations increase PITX2 activity. Phosphorylation of the PITX2 C-terminal tail facilitates protein interactions and increased transcriptional activity while phosphorylation of the N terminus inhibits PITX2 activity. We use a variety of PKC activators and inhibitors in cell cultures to demonstrate specific PITX2 activity regulated by PKC phosphorylation. ARS mutations have multiple effects on PITX2 phosphorylation by PKC. Conclusions: we demonstrate that PITX2 is specifically phosphorylated by PKC. Our data suggests that PKC plays an important role in tooth morphogenesis through the phosphorylation of PITX2 and regulation of its activity. Furthermore, we have identified several PITX2 mutations associated with ARS that affect phosphorylation and result in abnormal tooth development. Support for this research was provided from grant 1 RO1 DE13941 from the National Institute of Dental and Craniofacial Research.