Figure 4

Polo-like kinases can phosphorylate and disrupt the RENT complex in vitro. (A) Cdc5 can disrupt recombinant Net1N/Cdc14 complex. GST-Cdc14 was incubated with anti-T7 beads loaded with His6-T7-Net1N (assuming 100% binding efficiency, each reaction contained ~10 pmol of Cdc14 and ~80 pmol of Net1N), where Net1N consists of the N-terminal 341 amino acids of Net1. The beads were divided into equal portions, and treated with indicated amounts of Cdc5 (~10 pmol/μl). Proteins released into the supernatant (sup) or bound to the beads (bead) were fractionated by SDS-PAGE and immunoblotted with anti-T7 and anti-GST to detect His6-T7-Net1N and GST-Cdc14, respectively. (B) The Polo-like kinase Plx1 can also disrupt Net1N/Cdc14 complex. GST-T7-Cdc14 was captured on anti-GST resins, and incubated with His6-T7-Net1N. The resins were divided into two equal portions, and treated with either active (+) or inactive (m) Plx1. Proteins released from or bound to beads were fractionated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N. Note that phosphorylation by active Plx1 causes both GST-T7-Cdc14 and 1 + 156 - T7 - Metlin to migrate shower in SDS-Page. (C) Plx1 can disassemble immunoprecipitated RENT complex. RENT complex was retrieved from myc9-NET1 CDC14-HA3 cell lysate on a resin coated with 9E10 antibodies. The resin was divided into two equal portions, and treated with active (+) or inactive (m) Plx1. Proteins released into the supernatant or bound to the beads were separated by SDS-PAGE and immunoblotted with 9E10, 12CA5, and anti-Sir2 antibodies to detect Net1, Cdc14, and Sir2 proteins, respectively.