Protein kinase c phosphorylation modulates N- and C-terminal regulatory activities of the PTTX2 homeodomain protein
Protein kinase c phosphorylation modulates N- and C-terminal regulatory activities of the PTTX2 homeodomain protein
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DOI：
10.1021/bi048362x
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发表时间：
2005-03-15
期刊：
影响因子：
2.9
通讯作者：
Amendt, BA
中科院分区：
文献类型：
--
作者：
Espinoza, HM;Ganga, M;Amendt, BA
PKC磷酸化调节PITX2 DNA结合和转录活性。单个PKC位点的突变表明PITX2通过磷酸化进行功能调控。PITX2和PITX2 PKC突变蛋白的免疫沉淀揭示了转染细胞中PKC在体内的特异性磷酸化。PITX2的转录活性受n端磷酸化的负调控，受c端磷酸化的正调控。我们证明了通过PITX2 c端尾部磷酸化促进的蛋白质相互作用增加PITX2转录激活的机制。PITX2 C末端的磷酸化增强了与细胞因子的相互作用。与PITX2 PKC功能研究相证实，新发现的与Axenfeld-Rieger综合征（ARS）相关的c端PITX2突变显示磷酸化降低。该突变（PITX2 Delta T1261）在227密码子上产生移码突变，导致下游11个新的氨基酸，随后蛋白质过早截断，c端尾部和OAR结构域的3个PKC位点被删除，导致转录激活降低。PITX2 Delta T1261不能与细胞因子相互作用来协同激活转录，这证明了ARS与有缺陷的PITX2蛋白相互作用的第一个联系。纯合子Pitx2突变小鼠组织的基因表达谱显示，Dlx2表达降低是ARS患者相关发育缺陷的潜在分子基础。总的来说，磷酸化在发育过程中对PITX2同源结构域蛋白的活性进行了另一种水平的调节。
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 Delta T1261) 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 Delta T1261 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.