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Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.

Liu Y, Conaway L, Rutherford Bethard J, Al-Ayoubi AM, Thompson Bradley A, Zheng H, Weed SA, Eblen ST - Nucleic Acids Res. (2013)

Bottom Line: Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA.Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding.Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425, USA.

ABSTRACT
Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

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SPF45 overexpression enhances ovarian cancer cell migration in a phosphorylation site-specific manner. (A) SKOV-3 cell lines stably expressing Myc-SPF45, Myc-SPF45-8A, Myc-SPF45-8D or empty vector were grown to confluence. Cells were pre-treated with mitomycin-C for 3 h before being scratched and wound closure was recorded at 20 h by phase contrast microscopy. Representative images of six experiments are shown. (B) Wound closure in (A) was calculated using Image J software and expressed as a percentage of the initial scratched area. Results shown are mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group. (C and D) Scratch assays in OV2008 stable cells. The methods were the same as in (A and B). (E and F) Transwell migration assays. Stable (E) SKOV-3 cells (2 × 104) or (F) OV2008 cells (5 × 104 cells) in 0.1% FBS were added to the upper chamber, and 400 µl of 10% FBS was added to the lower chamber of a transwell dish. After 24 h, non-migrating cells were removed from the upper surface of the membrane, and the migrating cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrating cells were photographed and counted. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.
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gkt170-F9: SPF45 overexpression enhances ovarian cancer cell migration in a phosphorylation site-specific manner. (A) SKOV-3 cell lines stably expressing Myc-SPF45, Myc-SPF45-8A, Myc-SPF45-8D or empty vector were grown to confluence. Cells were pre-treated with mitomycin-C for 3 h before being scratched and wound closure was recorded at 20 h by phase contrast microscopy. Representative images of six experiments are shown. (B) Wound closure in (A) was calculated using Image J software and expressed as a percentage of the initial scratched area. Results shown are mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group. (C and D) Scratch assays in OV2008 stable cells. The methods were the same as in (A and B). (E and F) Transwell migration assays. Stable (E) SKOV-3 cells (2 × 104) or (F) OV2008 cells (5 × 104 cells) in 0.1% FBS were added to the upper chamber, and 400 µl of 10% FBS was added to the lower chamber of a transwell dish. After 24 h, non-migrating cells were removed from the upper surface of the membrane, and the migrating cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrating cells were photographed and counted. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.

Mentions: We previously showed that SPF45 overexpression affects cell proliferation and cell-matrix adhesion (21). To investigate the effect of SPF45 on cell motility, scratch wound healing assays were performed on SKOV-3 cells stably expressing Myc-SPF45, Myc-SPF45-8A, Myc-SPF45-8D or empty vector. Equal expression of Myc-SPF45 proteins was observed in the stable cell lines (Supplementary Figure S1). SPF45 and SPF45-8D significantly increased cell migration compared with vector control, whereas SPF45-8A did not (Figure 9A and B). Similar results were observed in OV2008 stable cell lines (Figure 9C and D). To confirm these results, we performed transwell cell migration assays towards a 10% serum chemoattractant. In SKOV-3 cells, SPF45 and SPF45-8D overexpression significantly increased serum-stimulated migration 2.1-fold and 2.2-fold, respectively, compared with vector control cells, whereas SPF45-8A inhibited cell migration by 41% (Figure 9E). In OV2008 cells, SPF45 and SPF45-8D enhanced migration by 3.2-fold and 3.4-fold, respectively, compared with vector control cells, whereas SPF45-8A had no effect (Figure 9F).Figure 9.


Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.

Liu Y, Conaway L, Rutherford Bethard J, Al-Ayoubi AM, Thompson Bradley A, Zheng H, Weed SA, Eblen ST - Nucleic Acids Res. (2013)

SPF45 overexpression enhances ovarian cancer cell migration in a phosphorylation site-specific manner. (A) SKOV-3 cell lines stably expressing Myc-SPF45, Myc-SPF45-8A, Myc-SPF45-8D or empty vector were grown to confluence. Cells were pre-treated with mitomycin-C for 3 h before being scratched and wound closure was recorded at 20 h by phase contrast microscopy. Representative images of six experiments are shown. (B) Wound closure in (A) was calculated using Image J software and expressed as a percentage of the initial scratched area. Results shown are mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group. (C and D) Scratch assays in OV2008 stable cells. The methods were the same as in (A and B). (E and F) Transwell migration assays. Stable (E) SKOV-3 cells (2 × 104) or (F) OV2008 cells (5 × 104 cells) in 0.1% FBS were added to the upper chamber, and 400 µl of 10% FBS was added to the lower chamber of a transwell dish. After 24 h, non-migrating cells were removed from the upper surface of the membrane, and the migrating cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrating cells were photographed and counted. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.
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Related In: Results  -  Collection

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gkt170-F9: SPF45 overexpression enhances ovarian cancer cell migration in a phosphorylation site-specific manner. (A) SKOV-3 cell lines stably expressing Myc-SPF45, Myc-SPF45-8A, Myc-SPF45-8D or empty vector were grown to confluence. Cells were pre-treated with mitomycin-C for 3 h before being scratched and wound closure was recorded at 20 h by phase contrast microscopy. Representative images of six experiments are shown. (B) Wound closure in (A) was calculated using Image J software and expressed as a percentage of the initial scratched area. Results shown are mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group. (C and D) Scratch assays in OV2008 stable cells. The methods were the same as in (A and B). (E and F) Transwell migration assays. Stable (E) SKOV-3 cells (2 × 104) or (F) OV2008 cells (5 × 104 cells) in 0.1% FBS were added to the upper chamber, and 400 µl of 10% FBS was added to the lower chamber of a transwell dish. After 24 h, non-migrating cells were removed from the upper surface of the membrane, and the migrating cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrating cells were photographed and counted. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.
Mentions: We previously showed that SPF45 overexpression affects cell proliferation and cell-matrix adhesion (21). To investigate the effect of SPF45 on cell motility, scratch wound healing assays were performed on SKOV-3 cells stably expressing Myc-SPF45, Myc-SPF45-8A, Myc-SPF45-8D or empty vector. Equal expression of Myc-SPF45 proteins was observed in the stable cell lines (Supplementary Figure S1). SPF45 and SPF45-8D significantly increased cell migration compared with vector control, whereas SPF45-8A did not (Figure 9A and B). Similar results were observed in OV2008 stable cell lines (Figure 9C and D). To confirm these results, we performed transwell cell migration assays towards a 10% serum chemoattractant. In SKOV-3 cells, SPF45 and SPF45-8D overexpression significantly increased serum-stimulated migration 2.1-fold and 2.2-fold, respectively, compared with vector control cells, whereas SPF45-8A inhibited cell migration by 41% (Figure 9E). In OV2008 cells, SPF45 and SPF45-8D enhanced migration by 3.2-fold and 3.4-fold, respectively, compared with vector control cells, whereas SPF45-8A had no effect (Figure 9F).Figure 9.

Bottom Line: Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA.Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding.Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425, USA.

ABSTRACT
Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

Show MeSH
Related in: MedlinePlus