Regulating transcriptional activity by phosphorylation: A new mechanism for the ARX homeodomain transcription factor.

Autor: Mattiske T; Adelaide Medical School, University of Adelaide, Adelaide, Australia.; Robinson Research Institute, University of Adelaide, Adelaide, Australia., Tan MH; Adelaide Medical School, University of Adelaide, Adelaide, Australia., Dearsley O; Adelaide Medical School, University of Adelaide, Adelaide, Australia.; Robinson Research Institute, University of Adelaide, Adelaide, Australia., Cloosterman D; Adelaide Medical School, University of Adelaide, Adelaide, Australia., Hii CS; Department of Immunopathology, SA-Pathology, Adelaide, Australia., Gécz J; Adelaide Medical School, University of Adelaide, Adelaide, Australia.; Robinson Research Institute, University of Adelaide, Adelaide, Australia.; Healthy Mothers and Babies, South Australian Health and Medical Research Institute, Adelaide, Australia., Shoubridge C; Adelaide Medical School, University of Adelaide, Adelaide, Australia.; Robinson Research Institute, University of Adelaide, Adelaide, Australia.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2018 Nov 12; Vol. 13 (11), pp. e0206914. Date of Electronic Publication: 2018 Nov 12 (Print Publication: 2018).
DOI: 10.1371/journal.pone.0206914
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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