ARX homeobox interacts with PICK1 and is phosphorylated by protein kinase C alpha

MH Tan1, O Dearsley1,2, C Moey1,2, CS Hii3, J Gecz1,2 and C Shoubridge1,2

  1. School of Medicine, University of Adelaide, SA 5005, Australia
  2. Robinson Research Institute, University of Adelaide, SA 5005, Australia
  3. Department of Immunopathology, SA-Pathology, SA 5006, Australia

Aristaless-related homeobox (ARX) gene is a homeodomain transcription factor important for the development of forebrain, pancreas and testes. Mutations in ARX result in intellectual disability with or without comorbidities of epilepsy and brain malformations. We show that ARX is phosphorylated and using mass spectrometry and in vitro kinase assays we identify three sites of phosphorylation. Additionally, using yeast-2-hybrid we identify a novel ARX-interacting protein, PICK1 (Protein interacting with C kinase 1). PICK1 interacts with the C-terminal region of ARX, confirmed by CoIP. PICK1 is a scaffold protein known to facilitate phosphorylation of protein partners by PRKCA (protein kinase C alpha). We confirm that ARX is phosphorylated by PRKCA using in vitro kinase assays and a specific PRKCA inhibitor. To interrogate the effect of phosphorylation on ARX function (ie transcriptional repression) we generated and tested phosphoablative mutants by replacing serines with alanines. These changes did not impact cellular localization nor transcriptional repressor activity as measured by dual-reporter luciferase assays (HEK293T cells). Currently we are using RNASeq of HEK293T and pancreatic alpha cells overexpressing wild-type and mutant ARX to identify the functional effects of ARX phosphorylation. In conclusion, our data indicates that PICK1 interacts with the C-terminal region of ARX, and this transcription factor is phosphorylated via PRKCA. Mutations in this region of ARX cause a severe early onset seizure phenotype in affected children. It is attractive to speculate that in these cases the function of this transcription factor is disrupted due to compromised post-translational regulation of phosphorylation.