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.
Carrier Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/metabolism , Nuclear Proteins/metabolism , Phosphorylation/physiology , Protein Kinase C-alpha/metabolism , Transcription Factors/metabolism , Animals , Cell Nucleus/metabolism , Gene Expression Profiling , Glucagon-Secreting Cells , HEK293 Cells , Homeodomain Proteins/genetics , Humans , Mice , Mutation , Serine/metabolism , Transcription Factors/genetics
PCR amplification (followed by mutation scanning or direct sequencing) is a technique widely used in mutation detection and molecular studies of disease-causing genes, such as ARX. PCR amplification of high GC-rich regions encounters difficulties using conventional PCR procedures. Here, we present the strategies to amplify and sequence these GC-rich regions for the purposes of mutation screening and other molecular analyses.
GC Rich Sequence , Homeodomain Proteins/genetics , Polymerase Chain Reaction/methods , Transcription Factors/genetics , DNA Mutational Analysis/methods , Humans
Co-immunoprecipitation (Co-IP) (followed by immunoblotting) is a technique widely used to characterize specific protein-protein interactions. Investigating interactions of proteins containing "sticky" polyalanine (PolyA) tracts encounters difficulties using conventional Co-IP procedures. Here, we present strategies to specifically capture proteins containing these difficult PolyA tracts, enabling subsequent robust detection of interacting proteins by Co-IP.
Homeodomain Proteins , Immunoprecipitation/methods , Peptides , Transcription Factors , Animals , Cell Line , Homeodomain Proteins/chemistry , Homeodomain Proteins/isolation & purification , Homeodomain Proteins/metabolism , Humans , Peptides/chemistry , Peptides/isolation & purification , Peptides/metabolism , Transcription Factors/chemistry , Transcription Factors/isolation & purification , Transcription Factors/metabolism
