Abstract
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.
Highlights
The Aristaless-related homeobox (ARX) gene [NM_139058.2] is a member of the paired-type homeodomain transcription factor family, with critical roles on development
Regulating transcriptional activity of the ARX homeodomain transcription factor by phosphorylation phosphorylation site(s) in ARX we designed seven partial ARX constructs spanning the fulllength protein (Fig 1A). When these partial recombinant ARX proteins were expressed in Human embryonic kidney 293T (HEK293T) cells and labelled as above, we determined a positive phosphorylation signal in the N-terminal octapeptide domain region (OP), polyalanine tract 1 & 2 region (PA1&2), polyalanine tract 3 region (PA3) and the homeodomain region (HD) (Fig 1C top panel— arrows)
We identified a novel ARX protein partner, protein interacting with C kinase 1 (PICK1), by yeast 2-hybrid and co-immunoprecipitation, which implicated protein kinase C alpha (PRKCA) as the ARX phospho-modifying enzyme
Summary
The Aristaless-related homeobox (ARX) gene [NM_139058.2] is a member of the paired-type homeodomain transcription factor family, with critical roles on development. Regulating transcriptional activity of the ARX homeodomain transcription factor by phosphorylation awarded to CS; Channel 7 Children’s Research Foundation (https://crf.org.au) grant 14916, awarded to CS; and the MS McLeod Foundation (Women’s and Children’s Hospital, Adelaide awarded to support a PhD Scholarship to MHT (http://www.wch.sa.gov.au/research/funding/ index_msmcleodpostgraduatescholarship.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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