Recent proteomic analyses suggest that ERj1 is a target of VX 950 [13] and [14]. Since ERj1 contains four potential phosphorylation motifs for the protein kinase CK2 in its cytosolic domain and because CK2 was previously found at the ER-membrane, we asked whether ERj1 is a CK2 substrate. Here we show that native ERj1 is phosphorylated in vitro by the CK2 holoenzyme. Using purified recombinant proteins, we detect a direct binding of CK2 to the cytosolic domain of ERj1 and a phosphorylation of the cytosolic domain of ERj1. A phosphorylation site mapping analysis showed that the phosphorylation site at amino acid residues 477–481 is the main target for CK2.
Materials and methods
Materials. Peroxidase conjugate of anti-rabbit IgG goat antibodies and thrombin were from Sigma Chemical Company. [γ32P]-ATP was from Hartmann Analytic. Enhanced chemiluminescence (ECL) and X-ray films were from GE Healthcare. PVDF membranes were from Millipore, CHAPS was from Calbiochem and λ-PPase was from New England Biolabs.