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, 41 (5), 436-443

Actin Dysfunction Induces Cell Cycle Delay at G2/M With Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

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Actin Dysfunction Induces Cell Cycle Delay at G2/M With Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

Deepmala Shrestha et al. Mol Cells.

Abstract

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was over-expressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was 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.

Keywords: Cdc25C and Wee1; G2/M delay; actin dysfunction; cytochalas-in D; extracellular signal-related kinase (ERK); human normal fibroblast IMR-90; ribosomal S6 kinase (RSK).

Figures

Fig. 1
Fig. 1. Actin disruption delays cell cycle at G2/M transition in normal IMR-90 cells
(A) The layout shows how the experiment was designed and performed. IMR-90 cells were synchronized with 2 mM double thymidine arrest. The cells were treated with 5 μM cytochalasin D (CD) or the solvent DMSO as a control at 5.5–6 h after the second release and were collected at each indicated time point after the second release. (B) CD-treated and CD-untreated control cells were immunostained with an antibody against α-tubulin as described in the Materials and Methods. Nuclei were counterstained with DAPI. Scale bar, 50 μm. (C) Cells were fixed, stained with PI (10 μg/ml), and processed for flow cytometry. For each analysis, 10,000 cells were counted, and each fraction of cells with 2N and 4N DNA content is shown. (D) The cell lysate was resolved by 8% SDS-PAGE and blotted with anti-cyclin A and reprobed with anti-actin. (E) Histone extraction was performed as described in the “Materials and Methods”. The cytosolic fraction and histone fraction was resolved by 12% SDS-PAGE and blotted with p-Cdc2 (Tyr 15) and p-H3 Ser 10, respectively. Actin and histone H3 were used as loading controls.
Fig. 2
Fig. 2. Actin dysfunction sustains RSK activation and Cdc2 inactivation in IMR-90 cells
As denoted in Fig. 1A, IMR-90 cells were synchronized with 2 mM double thymidine arrest, incubated with 5 μM cytochalasin D or the solvent DMSO as a control at 5.5–6 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 of Cdc2. (C) The same samples from (A) and (B) were blotted with p-Wee1 (Ser 642) and re-probed with anti-Wee1. Each blot was re-probed with anti-actin as a loading control.
Fig. 3
Fig. 3. Overexpression of dominant-negative RSK partially releases the cell cycle delay by actin dysfunction
(A) The layout shows how the experiment was performed. IMR-90 cells were synchronized with 2 mM thymidine and transfected with DN-RSK or empty vector as described in the Materials and Methods. Cells were treated with 5 μM CD or only DMSO as a control at 5.5–6 h after the second release and collected at each indicated time point after the second release. Each cell lysate was resolved by 8% SDS-PAGE and blotted with (B) anti-FLAG and anti-actin, (C) p-ERK1/2 and p-Cdc2 (Tyr 15), (D) p-Cdc25C (Ser 216), and p-Wee1 (Ser 642). Each membrane was reprobed with anti-actin as a loading control.
Fig. 4
Fig. 4
A schematic model representing the mechanism to delay the cell cycle at G2/M by actin disruption.

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