Fig. 4.

The Mos/MAPK/Rsk pathway promotes interaction of Emi2 with PP2A. (A) CSF extracts were preincubated for 5 min with 0.5 mg/ml GST or GST-Emi2 (amino acids 319–375, SD), after which IVT ³⁵S-labeled Emi2 was added to the extract. Samples were taken at indicated times to detect cyclin B2 and added GST by immunoblotting, Cdc2 kinase activity, and full-length Emi2 tracer by autoradiography. In addition, samples were taken to examine Hoechst-stained nuclei supplemented into the extract. (Scale bars: 25 μm.) (B) MBP or MBP-Emi2N (amino acids 1–350) prebound to amylose resin was added to CSF extracts in the presence of DMSO or 250 μM U0126. After 30 min of incubation at room temperature, the beads were washed, and bound PP2A was examined by immunoblotting. (C) Indicated GST-SD proteins prebound to glutathione Sepharose were incubated in interphase extracts pretreated with MBP or Mos protein. After incubation, beads were retrieved and washed, and bound PP2A was examined by immunoblotting. (D) Indicated GST-SD prebound to glutathione Sepharose was phosphorylated in vitro by using recombinant Rsk and [γ-³²P]ATP. After washing, proteins were separated by SDS/PAGE. Proteins were Coomassie blue stained, and incorporated ³²P was detected by autoradiography. (E) Indicated GST-SD proteins were incubated in CSF extracts and handled as in B. (F) Indicated radiolabeled IVT Emi2 was added to CSF extracts and incubated at room temperature. Samples were taken at indicated times, and levels of ³⁵S-labeled Emi2 were examined by SDS/PAGE and autoradiography. (G) Indicated radiolabeled IVT Emi2 were mixed into CSF extracts. After 2 h of incubation at 4°C, Cdc27 was immunoprecipitated and washed, and bound IVT Emi2 was examined by SDS/PAGE and autoradiography. (H) Mos-activated Rsk phosphorylates Emi2 at S335 and T336. This phosphorylation promotes Emi2–PP2A association, enhancing Emi2 dephosphorylation at both N- and C-terminal Cdc2 phosphorylation sites. Dephosphorylation of the N-terminal sites promotes Emi2 stability. Dephosphorylation of the C-terminal sites enhances APC-inhibitory binding of Emi2.