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First published online 29 March 2006
doi: 10.1242/dev.02348

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p90^Rsk is required for G1 phase arrest in unfertilized starfish eggs

Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan.

View larger version (30K): [in a new window]   Fig. 1. Rsk is activated downstream of Mos-MAPK pathway in starfish oocytes. (A) Specificity of antibodies against the N-terminal kinase domain of starfish Rsk. Lysates from immature oocytes were separated on a 10% SDS-PAGE gel and blots were probed with pre-immune sera and anti-Rsk sera (1 and 2), respectively. Right side, molecular mass markers in kDa. (B) Dynamics of Rsk during starfish meiotic cycles. Extracts were prepared from oocytes and fertilized eggs at 10 minutes intervals after 1-MeAde addition, and immunoblotted with anti-MAPK antibody (second panel) and anti-Rsk serum 2 (third panel). Immunoprecipitates with the anti-Rsk serum 1 were assayed for phosphorylation of GST-S6. Rsk activity was detected on an autoradiogram (fourth panel), which was then quantified (fifth panel). Extracts were also assayed for phosphorylation of histone H1 (first panel). U, M and L indicate three forms of Rsk. The upper and lower bands of MAPK correspond to the active and inactive forms, respectively. Arrows and arrowheads indicate the time of GVBD and fertilization, respectively. (C) Activation of MAPK and Rsk by GST-Mos injection. Immature oocytes were injected with 25 pg of control GST (left) or GST-Mos (right), recovered at 60 minutes, and immunoblotted with anti-Rsk serum 2 (upper) and anti-MAPK antibody (lower). (D) Rsk inactivation by MAPK inactivation. Maturing oocytes 50 minutes after 1-MeAde addition were treated with 10 µM U0126, an inhibitor of MAPK kinase, or control DMSO, and then recovered at 10-minutes interval. The extracts were immunoblotted with anti-Rsk serum 2 (upper) and anti-MAPK antibody (lower).

View larger version (23K): [in a new window]   Fig. 2. Anti-Rsk antibody 2 neutralizes Rsk activity in vitro and in vivo. (A) The anti-Rsk serum 2 inhibits Rsk activity in vitro. Extracts were prepared from immature oocytes (0) and unfertilized mature eggs (150). Immunoprecipitates with anti-Rsk serum 1 and 2 from these extracts were immunoblotted with anti-Rsk serum 2 (upper), or assayed for phosphorylation of S6 peptide (lower). (B) The purified anti-Rsk antibody 2 inhibits Rsk activity in vivo. Immature oocytes were injected with 3 ng of the anti-Rsk antibody purified from the serum 2 (lane 6) or control IgG from preimmune serum 2 (lane 5), or not injected (lane 1-4); oocytes were then treated with 1-MeAde. Extracts were prepared at the indicated times after 1-MeAde addition, and immunoblotted with anti-MAPK antibody (third panel) and anti-active MAPK (fourth panel). Immunoprecipitates with the anti-Rsk serum 1 were immunoblotted with anti-Rsk serum 2 (second panel), and assayed for phosphorylation of GST-S6 (first panel). In lane 4 (210F), mature eggs were inseminated 150 minutes after 1-MeAde addition. In lane 6, Rsk was present in the U form but its activity was almost undetectable.

View larger version (49K): [in a new window]   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.

View larger version (72K): [in a new window]   Fig. 4. Mos causes G1 arrest in starfish blastomeres. (A,B) GST-Mos injection activates MAPK and Rsk, and causes cell cycle arrest at interphase in blastomeres. Mature eggs 2.5 hours after 1-MeAde addition were inseminated. At the two-cell stage (around 1.5-2.0 hours after insemination), one cell was injected with 25 pg of GST-Mos or control GST, or not injected, and the embryos were further cultured for 5-5.5 hours. At indicated times, lysates from whole embryos were immunoblotted with anti-MAPK antibody, or immunoprecipitated with anti-Rsk serum 1 for phosphorylation of GST-S6 (A). Whereas the non-injected half developed to the early blastula stage, nuclei were observed in the GST-Mos-injected half of large cells, indicating the cell cycle arrest at interphase (B, right). (C) GST-Mos causes G1 arrest after fertilization. Mature eggs were inseminated, and 20-30 minutes later (around entry into the first S-phase), were injected with GST-Mos or control GST, or not injected. After an additional 30 minutes (around entry into the first M-phase in control eggs), eggs were incubated for 3 hours in the presence of BrdU to examine DNA replication. In control eggs injected with GST, although significant delay in cell cycle progression was observed, BrdU was clearly incorporated into DNA. By contrast, no incorporation of BrdU was seen in GST-Mos-injected eggs, indicating cell cycle arrest at G1 phase. The numbers of eggs examined are indicated in parentheses.

View larger version (61K): [in a new window]   Fig. 5. Rsk is necessary for preventing parthenogenetic activation after meiosis I in starfish oocytes. (A,B) Emission of only one polar body from oocytes lacking Rsk activity, followed by parthenogenesis. Immature oocytes were injected with 3 ng of control IgG or the neutralizing anti-Rsk antibody 2, and then treated with 1-MeAde. These oocytes were either fixed at 2.5 hours after 1-MeAde addition, followed by DNA staining with DAPI (A), or further incubated in the absence of insemination (B). The numbers of eggs examined are indicated in parentheses. Arrowheads indicate polar bodies and arrows indicate nucleus (A). Photographs of blastomere and gastrula were taken 7 and 22 hours, respectively, after 1-MeAde addition (B). (C) Dynamics of H1 kinase activity in oocytes lacking Rsk activity. Extracts were prepared at 15-minute intervals after 1-MeAde addition from oocytes that had been injected with 3 ng of control IgG or the neutralizing anti-Rsk antibody 2. Histone H1 kinase activity was detected on an autoradiogram (upper), which was then quantified (lower). (D) Dynamics of phosphorylation states of meiotic cell cycle regulators in oocytes lacking Rsk activity. Lysates from oocytes, that had been injected with control IgG or the neutralizing anti-Rsk antibody 2, were prepared at 15-minute intervals after 1-MeAde addition, and immunoblotted with anti-Rsk serum 2, anti-Myt1, anti-Cdc25, anti-MAPK, anti-active MAPK, anti-phospho Tyr-Cdc2 antibodies.

View larger version (21K): [in a new window]   Fig. 6. Meta-II-CSF versus G1-CSF. The Mos-MAPK pathway functions as `meta-II-CSF' in frog and mouse eggs, and as `G1-CSF' in starfish eggs, awaiting fertilization. Rsk is an essential component for G1-CSF, but not for meta-II-CSF.

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