Figure 2. SRPK2 phosphorylates acinus on Serine 422.

(A). Diagram of Acinus-S. Acinus-S possesses three RS motifs as indicated. The three fragments with each containing the RS dipeptide repeat motif are indicated with residue numbers. (B). In vitro SRPK2 kinase assay. Purified recombinant GST-fusion proteins were incubated with purified His-SRPK2 at 30 °C for 30 min. Both fragments B and C were robustly phosphorylated, while fragment A was not (left panel). S422 residue in acinus-S was phosphorylated by SRPK2. Purified GST-acinus proteins were incubated with purified SRPK2 in the presence of [γ-³²P]-ATP. S422A mutant substantially decreased acinus phosphorylation (middle and right panels). (C). Wild-type but not kinase-dead SRPK2 phosphorylates acinus. GST-acinus wt and S422A were transfected into HEK293 cells in the presence or absence of SRPK2. Transfected acinus was pulled down with glutathione beads. While S422 site was markedly phosphorylated in wild-type acinus, no phosphorylation was detected in S422A mutant (top left panel). The expression of transfected constructs was verified (2^nd to bottom left panels). Flag-acinus and Myc-SRPK2 wt or KD were transfected into HEK293 cells. Acinus was immunoprecipitated with anti-Flag antibody and probed with anti-phospho-S422 antibody. Wild-type SRPK2 potently phosphorylated acinus, whereas SRPK2-KD failed (top right panel). The expression of transfected constructs was verified (2^nd to bottom right panels). (D). Acinus-S can be phosphorylated in intact cells. HEK293 cells were transfected with si-RNA for SRPK2 or Akt, and followed by serum starvation overnight. In control samples, serum triggered potent S422 phosphorylation. Knocking down of SRPK2 or Akt blocked acinus S422 phosphorylation (top panel). The depletion of SRPK2 and Akt was confirmed (2^nd and 3^rd panels).