As expected, RSK activation was sustained after CD treatment in normal IMR-90 cells (Fig. was Sulfo-NHS-Biotin maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1. egg extracts (Chun et al., 2005). We then questioned whether ERK activation by actin disruption activates RSK downstream of ERK1/2 in IMR-90 cells, leading to Cdc2 inhibition to cause G2/M delay. First, we examined the Sulfo-NHS-Biotin activation of RSK downstream of ERK1/2 by actin dysfunction in IMR-90 cells. The expression levels of ERK1/2, RSK1, and Cdc2 were similar in both CD-treated and untreated IMR-90 cells (Figs. 2A and 2B). As reported by Lee and Song (2007), ERK activation was sustained for 30C60 min in CD-treated cells (Figs. 2A and 2B). Consistent with sustained ERK activation, continued activation of RSK1 was observed in IMR-90 cells treated with CD (Fig. 2A). In addition, inhibitory phosphorylation of Cdc2 (Tyr 15) was maintained until 10.5 h after the release in CD-treated IMR-90 cells, while it started to decline between 9C9.5 h in CD-untreated control cells, supporting G2/M delay of the cell cycle (Figs. 2A and 2B). Taken together, these observations demonstrate that actin dysfunction sustains RSK1 activation concomitantly with ERK activation and delays the cell cycle at G2/M by inhibiting Cdc2 kinase in normal Sulfo-NHS-Biotin IMR-90 cells. Open in a separate window Fig. 2 Actin dysfunction sustains RSK activation and Cdc2 inactivation in IMR-90 cellsAs denoted in Fig. 1A, IMR-90 cells were synchronized with 2 mM double thymidine arrest, incubated with 5 M cytochalasin D or the solvent DMSO as a control at 5.5C6 h after the second release, and collected at each indicated time point after the second release. Cell lysates were resolved by 8% SDS-PAGE and blotted. Blots were probed with (A) p-ERK1/2 and p-RSK1 (Ser 380) and re-probed with anti-ERK1/2 and anti-RSK1 to observe the total amount of each protein, (B) p-ERK1/2 and p-Cdc25C (Ser 216), and re-probed with anti-ERK1/2 and anti-Cdc25C. (A, B) Cell cycle progression at G2/M was monitored by detecting p-Cdc2 (Tyr 15) followed by re-probing with anti-Cdc2 to detect the total amount Sulfo-NHS-Biotin of Cdc2. (C) The same samples from (A) and (B) were blotted with p-Wee1 (Ser 642) and re-probed with Sulfo-NHS-Biotin anti-Wee1. Each blot was re-probed with anti-actin as a loading control. In CD-treated IMR-90 cells, we observed that the inhibitory phosphorylation of Cdc2 (Tyr 15) was maintained until 10.5 h after release (Figs. 2A and 2B). It is well-known that Wee1 inactivates Cdc2 kinase by phosphorylating Rabbit Polyclonal to KLF11 Tyr 15, which is removed by Cdc25C phosphatase to activate Cdc2. Thus, we examined how actin dysfunction by CD controls Cdc25C and Wee1 to inhibit the kinase activity of Cdc2 to cause G2/M delay. Cdc25C activity is controlled by inhibitory phosphorylation at Ser 216, which is mainly detected during interphase (Peng et al., 1997). Once the cell enters mitosis, Ser 216 of Cdc25C is dephosphorylated and activating phosphorylation of Cdc25C at Ser 214 is detected during mitosis (Bulavin et al., 2003; Peng et al., 1997). Inhibitory phosphorylation of Cdc25C at Ser 216 in CD-treated IMR-90 cells was maintained.