Figure 4.

Ser-209 and Thr-210 are major BSK5 sites phosphorylated in vitro by PEPR1 and EFR. BSK5, BSK5^S209A, BSK5^T210A, BSK5^S209A/T210A, and the kinase domain (KD) of PEPR1 and EFR fused to GST were expressed in E. coli and purified. Phosphorylation of GST-BSK5, GST-BSK5^S209A, GST-BSK5^T210A, and GST-BSK5^S209A/T210A by GST-PEPR1-KD (A) and GST-EFR-KD (B) was assayed in vitro in the presence of [γ-³²P]ATP. Proteins were fractionated and either transferred to a PVDF membrane and exposed to autoradiography or stained with Coomassie Blue. Experiments were repeated three times with similar results.