FIGURE 4.

B55β regulates CaMKII activation. A, CaM-Sepharose was incubated with Xenopus egg extracts treated with or without G6P and incubated for 1 h at 4 °C. Beads were retrieved by centrifugation and analyzed by immunoblotting for PP2A regulatory subunits. B, GST-B55β or GST bound to glutathione-Sepharose was incubated for 1 h at 4 °C with Xenopus egg extracts treated with or without G6P. Beads were retrieved by centrifugation and analyzed for CaMKII or B55β immunoblotting. Note that the top and middle panels are from two different films with different exposures as the CaMKII antibody recognized CaMKIIα much more strongly than the other isoforms. C, Xenopus CaMKIIγ WT protein expressed from baculoviral vectors in SF9 cells was added into Xenopus egg extracts for 0.5 h at room temperature, and then the extracts were treated with or without G6P for 0.5 h at room temperature and incubated with GST-B55β or GST bound to glutathione-Sepharose for 1 h at 4 °C. The beads were retrieved by centrifugation and analyzed for CaMKII or B55β immunoblotting. D, B55β antibody or control rabbit IgG bound to Dynabead-linked protein A was incubated with Xenopus egg extracts for 1 h at 4 °C and removed with a magnet (repeated three times). Extracts were analyzed by B55β and PP2A C subunit immunoblotting. E, the B55β-depleted or undepleted extract was treated with or without G6P for 0.5 h and analyzed by pT286 immunoblotting. F, GST tagged Xenopus caspase-2 prodomain or GST bound to glutathione-Sepharose was incubated with B55β-depleted or undepleted Xenopus egg extracts supplemented with [γ-³²P]ATP and treated with or without G6P. The samples were resolved by SDS-PAGE and detected by autoradiography. CB, Coomassie Blue. G, B55β-specific or control siRNA-treated 293T cells were glucose-starved for 12 h and then incubated with or without 25 mm glucose. The lysates were analyzed by immunoblotting for pT286, CaMKII, and B55β.