Figure 1.

Dual specificity tyrosine-phosphorylation-regulated kinase 1A interacts with GluN2A and phosphorylates the GluN2A subunit at S¹⁰⁴⁸. (A) Solubilized proteins from the adult mouse brain (input lane; 10% of lysates) were immunoprecipitated with either a mouse IgG or an anti-DYRK1A antibody, and both the lysates and the immunoprecipitates were analyzed in Western blots probed with anti-GluN2A and anti-DYRK1A antibodies as indicated. (B) Equivalent aliquots of anti-GluN1 purified immunocomplexes obtained from the adult mouse brain were analyzed in Western blots probed with an anti-GluN2A antibody (left panel) or they were used as the substrate in a radioactive in vitro phosphorylation assay in the presence or absence of purified recombinant GST-DYRK1A. The radiolabeled proteins were then fractionated by SDS-PAGE and detected by autoradiography (right panel). The arrows indicate the phosphorylated GluN2A and possibly, the phosphorylated GluN1, and the stars indicate the labeled bands resulting from GST-DYRK1A autophosphorylation. (C) Schematic representation of the GluN2A subunit topology and the GST fusion proteins covering the cytoplasmic tail of GluN2A used in the assays. (D) As indicated, bacterially purified GluN2A GST-fusions or unfused GST were examined in an IVK assay, in the presence of a GST fusion protein of the wild-type (wt) or kinase-deficient DYRK1A (KD). The substrates were analyzed by Coomassie staining (left panel) and the phosphorylated bands indicated by black arrows represent the full-length recombinant proteins, while the white arrows refer to the GluN2ACter truncated products and the asterisks indicate the GST-DYRK1Awt autophosphorylated bands (full-length or truncated products). (E) Schematic representation of the different mutant variants of the GST-GluN2AC1 fragment in which the asterisks indicate the position of the corresponding Ser to Ala mutants. (F) The GST-GluN2AC1 fragment or the indicated mutants were used as substrates in IVK assays with GST-DYRK1A. The panel shows a representative experiment and the histogram corresponds to the average ³²P incorporation ± SEM (n = 2–3) calculated by densitometry (*p < 0.05). (G) The amino acid sequences of GluN2A from human (NP_000824), mouse (NP_032196), rat (NP_036705), dog (XP_005621613), sheep (XP_004020812) and chicken (XP_425252) were aligned to show the conserved region surrounding S¹⁰⁴⁸ (marked with an asterisk). The numbers indicate the first and last amino acids listed.