Adelaide Medical School, University of Adelaide, Adelaide, Australia.
Robinson Research Institute, University of Adelaide, Adelaide, Australia.
PLoS One. 2018 Nov 12;13(11):e0206914. doi: 10.1371/journal.pone.0206914. eCollection 2018.
Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in development. Here we identify that ARX protein is phosphorylated. Using mass spectrometry and in vitro kinase assays we identify phosphorylation at serines 37, 67 and 174. Through yeast-2-hybrid and CoIP we identified PICK1 (Protein interacting with C kinase 1) binding with the C-terminal region of ARX. PICK1 is a scaffold protein known to facilitate phosphorylation of protein partners by protein kinase C alpha (PRKCA). We confirm that ARX is phosphorylated by PRKCA and demonstrate phosphorylation at serine 174. We demonstrate that phosphorylation is required for correct transcriptional activity of the ARX protein using transcriptome-wide analysis of gene expression of phospho-null mutants (alanines replacing serines) compared to ARX wild-type (ARX-WT) overexpressed in pancreatic alpha TC cells. Compared to untransfected cells, ARX-WT overexpression significantly altered expression of 70 genes (Log2FC >+/-1.0, P-value <0.05). There were fewer genes with significantly altered expression compared to untransfected cells with the double phospho-null mutant Ser37Ala+Ser67Ala (26%) and Ser174Ala (39%), respectively. We demonstrate that the c-terminal region of ARX required to bind PICK1 causes a shift in PICK1 subcellular localisation to the nucleus to co-locate with the ARX protein, and truncation of this C-terminal region leads to the same loss of transcriptional activation as S174A mutant. In conclusion, we show that ARX is phosphorylated at several sites and that this modification affects its transcriptional activity.
Aristaless 相关同源盒（ARX）基因编码一种配对型同源结构域转录因子，在发育过程中具有关键作用。在这里，我们发现 ARX 蛋白发生磷酸化。通过质谱分析和体外激酶实验，我们鉴定到 ARX 蛋白在丝氨酸 37、67 和 174 位点发生磷酸化。通过酵母双杂交和 CoIP 实验，我们发现 PICK1（与蛋白激酶 Cα相互作用的蛋白 1）与 ARX 的 C 端区域结合。PICK1 是一种已知的支架蛋白，可促进蛋白激酶 Cα（PRKCA）对蛋白伴侣的磷酸化。我们证实 ARX 可被 PRKCA 磷酸化，并证明丝氨酸 174 发生磷酸化。我们通过对胰腺αTC 细胞中过表达 ARX 野生型（ARX-WT）和磷酸化缺失突变体（丝氨酸突变为丙氨酸）的转录组范围的基因表达进行全基因组分析，证实了磷酸化对于 ARX 蛋白的正确转录活性是必需的。与未转染的细胞相比，ARX-WT 的过表达显著改变了 70 个基因的表达（Log2FC >+/-1.0，P 值 <0.05）。与未转染的细胞相比，双磷酸化缺失突变体 Ser37Ala+Ser67Ala（26%）和 Ser174Ala（39%）的基因表达发生显著改变的基因更少。我们证明 ARX 的 C 端区域与 PICK1 结合，导致 PICK1 的亚细胞定位发生核转位，与 ARX 蛋白共定位，并且该 C 端区域的缺失会导致与 S174A 突变体相同的转录激活丧失。总之，我们表明 ARX 在多个位点发生磷酸化，并且这种修饰会影响其转录活性。
