Protein Kinase C Phosphorylation Modulates N- and C-Terminal Regulatory Activities of the PITX2 Homeodomain Protein

Abstract

PKC phosphorylation regulates PITX2 DNA binding and transcriptional activity. Mutation of individual PKC sites demonstrates the functional regulation of PITX2 through phosphorylation. Immunoprecipitation of PITX2 and a PITX2 PKC mutant protein reveal specific in vivo phosphorylation by PKC in transfected cells. The transcriptional activity of PITX2 is negatively regulated by N-terminal phosphorylation and positively regulated by C-terminal phosphorylation. We demonstrate a mechanism of increased PITX2 transcriptional activation through protein interactions facilitated by phosphorylation of the PITX2 C-terminal tail. Phosphorylation of the PITX2 C terminus enhances the interaction with cellular factors. In corroboration with the PITX2 PKC functional studies, a newly identified C-terminal PITX2 mutation associated with Axenfeld-Rieger syndrome (ARS) demonstrates reduced phosphorylation. This mutation (PITX2 DeltaT1261) creates a frameshift mutation in codon 227 resulting in 11 novel amino acids downstream followed by premature truncation of the protein. Three PKC sites in the C-terminal tail and OAR domain are deleted, which results in decreased transcriptional activation. PITX2 DeltaT1261 is unable to interact with a cellular factor to synergistically activate transcription and demonstrates the first link of ARS with defective PITX2 protein interactions. Gene expression profiling of homozygous Pitx2 mutant mouse tissue reveals decreased Dlx2 expression as a potential molecular basis for developmental defects associated with ARS patients. Overall, phosphorylation imparts another level of regulation to the activity of the PITX2 homeodomain protein during development.

Molecular Consequences of a Frameshifted DLX3 Mutant Leading to Tricho-Dento-Osseous Syndrome

“…AxenfeldRieger syndrome is an autosomal dominant human disorder characterized by ocular anterior chamber anomalies, dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities (31). Frameshift and nonsense mutations occurring in the PITX2 C-terminal domain have been detailed (25,26). The truncated isoform ⌬T1261 of the PITX2a gene is able to bind DNA but is unable to interact with its partner Pit-1 thus exhibits a reduced transactivation capacity (25).…”

“…Frameshift and nonsense mutations occurring in the PITX2 C-terminal domain have been detailed (25,26). The truncated isoform ⌬T1261 of the PITX2a gene is able to bind DNA but is unable to interact with its partner Pit-1 thus exhibits a reduced transactivation capacity (25). A second study described a deletion that results in a frameshift starting in codon 122 (D122FS) and in a truncated protein of 153 amino acids (26), and a previously described nonsense mutation that introduces a stop codon in position 133 (W133Stop).…”

Hierarchical Interactions of Homeodomain and Forkhead Transcription Factors in Regulating Odontogenic Gene Expression

“…To test the significance of Pitx2 and Dlx2 binding to the Dlx2 promoter, transient transfection assays were performed in CHO cells. PITX2A is a full-length construct, and PITX2A ⌬N38 is a N-terminal deletion construct, whereas the PITX2A ⌬T1261 is an Axenfeld-Rieger syndrome mutation and has a stop codon in the C-terminal OAR domain (29) (Fig. 3A).…”

Molecular Cloning of Paired Related Homeobox 2 (Prx2) as a Novel Pituitary Transcription Factor

“…The C-terminal element of Ptx2 plays important roles in gene regulation by which Pit1 interacts with the C-terminal tail of Ptx2 to disrupt its interference with DNA binding ability [47]. Phosphorylation of the C-terminal tail of Ptx2 reportedly enhances the interaction with cellular factors [49]. If the OAR of Prx2 has a role similar to the C-terminal of Ptx1 and/or Ptx2, Prx2 may be involved in regulation by protein-protein interaction.…”