Fig. 4. cyclin B/CDC2 and MAP kinase phosphorylate DOC1R. (A) Purified cyclin B/CDC2 and MAPK phosphorylate DOC1R in vitro. Purified cyclin B/CDC2 (lanes 1, 2 and 3) and recombinant active rat ERK2 (lanes 4, 5 and 6) were incubated with (+) or without (-) 6His-DOC1R or Histone H1 (lanes 2 and 5) in the presence of [-³²P]-ATP. The [³²P] incorporation was detected by autoradiography. This experiment has been repeated twice. (B) The MOS/.../MAPK pathway phosphorylates DOC1R. MYC-DOC1R-injected oocytes from wild-type (lane 1) or Mos^-/- (lane 2) mice were cultured for 12 hours after GVBD and collected. This experiment has been repeated three times. (C) MYC-DOC1R expression after microinjection of RNA encoding MYC-DOC1R into immature wild-type or Mos^-/- oocytes. Forty wild-type oocytes cultured for 12 hours after GVBD (top panel) or 40 Mos^-/- oocytes cultured for 12 hours (bottom panel) after GVBD were collected and analysed by 2D gel electrophoresis. The migration of MYC-DOC1R is shifted towards the acidic pole (H+) in wild-type oocytes compared with its migration in Mos^-/- oocytes. To position the different MYC-DOC1R isoforms from one sample to the other, we re-probed all the blots using Ezrin as an internal control (Louvet-Vallee et al., 2001). This experiment has been repeated three times. Samples in B and C were analyzed by immunoblotting using an anti-MYC antibody.