Fig. 3. Rsk is necessary and sufficient for G1 arrest in starfish eggs. (A) Inhibition of Rsk activity causes DNA replication. Unfertilized mature eggs after completion of meiosis II (120 minutes after 1-MeAde addition) were injected in the presence of BrdU with 3 ng of either the neutralizing anti-Rsk antibody 2, the control IgG from preimmune serum 2 or the control blocked anti-Rsk antibody 2. Some eggs that received the injection of the neutralizing anti-Rsk antibody 2 were further injected with 0.6 ng of CA-Rsk-EE or CA-Rsk-EE KD. Thereafter, eggs were fixed at 60 minutes incubation. DNA replication was detected by incorporation of BrdU (top panels). Lysates from these eggs were immunoblotted with anti-MAPK antibody (bottom panels). Another aliquots of the lysates were directly processed (bottom right; S6 kinase activity) or immunoprecipitated with anti-Rsk serum 1 (bottom left; Rsk activity) for phosphorylation of GST-S6. MAPK remained in the active form, even though BrdU was incorporated. As a standard, the states of MAPK, Rsk activity and S6 kinase activity were shown in mature eggs incubated for 60 minutes without (-) or with (+) fertilization. (B) Maintenance of Rsk activity prevents DNA replication. Mature eggs after completion of meiosis II were injected with 0.6 ng of CA-Rsk-EE or control CA-Rsk-EE KD, or not injected. These eggs were inseminated in the presence of BrdU, and were fixed after 50 minutes incubation. DNA replication was detected by incorporation of BrdU (left and middle). Lysates from these eggs were immunoblotted with anti-MAPK antibody (right, upper), or assayed for total S6 kinase activity (right, middle). For assay of Rsk activity by phosphorylation of GST-S6, Rsk was precipitated with anti-serum 1 for control non-injected eggs, or with glutathione-Sepharose 4B for CA-Rsk-EE or CA-Rsk-EE KD-injected eggs (right, lower). In CA-Rsk-EE-injected and inseminated eggs, MAPK was converted to the inactive form, supporting successful insemination even though DNA replication was almost undetectable. In both A and B, the numbers of eggs examined are indicated in parentheses. In the fused nucleus (B), the bright area corresponds to DNA derived from sperm nucleus, while the rest is from egg nucleus.