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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 invasion in a phosphorylation site-specific manner. (A) Stable SKOV-3 cells (2 × 104) and (B) OV2008 (5 × 104) cells in 0.5 ml of 0.1% FBS were added to the top of a matrigel invasion chamber, with 0.75 ml of 10% FBS in the lower chamber. After 48 h, the non-invasive cells were removed from the upper surface of the membrane, and the invading cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Representative pictures of invading cells are shown. (C and D) Quantification of cell invasion from (C) SKOV-3 and (D) OV2008 stable cell lines in (A and B) from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group.
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gkt170-F10: SPF45 overexpression enhances ovarian cancer cell invasion in a phosphorylation site-specific manner. (A) Stable SKOV-3 cells (2 × 104) and (B) OV2008 (5 × 104) cells in 0.5 ml of 0.1% FBS were added to the top of a matrigel invasion chamber, with 0.75 ml of 10% FBS in the lower chamber. After 48 h, the non-invasive cells were removed from the upper surface of the membrane, and the invading cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Representative pictures of invading cells are shown. (C and D) Quantification of cell invasion from (C) SKOV-3 and (D) OV2008 stable cell lines in (A and B) from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group.

Mentions: In addition, we determined the effect of SPF45 overexpression on cell invasion through matrigel in a transwell chamber. In SKOV-3 cells, SPF45 and SPF45-8D significantly increased cell invasion by 1.9-fold and 2.1-fold, respectively, compared with vector control cells, whereas 8A-SPF45 inhibited cell invasion by 45% (Figure 10A and C). In OV2008 cells, SPF45 and SPF45-8D enhanced invasion by 3.2-fold and 3.5-fold, respectively, whereas SPF45-8A did not significantly affect invasion (Figure 10B and D). Collectively, these results show that SPF45 overexpression promoted cell migration and invasion in a phosphorylation-dependent manner.Figure 10.


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 invasion in a phosphorylation site-specific manner. (A) Stable SKOV-3 cells (2 × 104) and (B) OV2008 (5 × 104) cells in 0.5 ml of 0.1% FBS were added to the top of a matrigel invasion chamber, with 0.75 ml of 10% FBS in the lower chamber. After 48 h, the non-invasive cells were removed from the upper surface of the membrane, and the invading cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Representative pictures of invading cells are shown. (C and D) Quantification of cell invasion from (C) SKOV-3 and (D) OV2008 stable cell lines in (A and B) from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643583&req=5

gkt170-F10: SPF45 overexpression enhances ovarian cancer cell invasion in a phosphorylation site-specific manner. (A) Stable SKOV-3 cells (2 × 104) and (B) OV2008 (5 × 104) cells in 0.5 ml of 0.1% FBS were added to the top of a matrigel invasion chamber, with 0.75 ml of 10% FBS in the lower chamber. After 48 h, the non-invasive cells were removed from the upper surface of the membrane, and the invading cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Representative pictures of invading cells are shown. (C and D) Quantification of cell invasion from (C) SKOV-3 and (D) OV2008 stable cell lines in (A and B) from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group.
Mentions: In addition, we determined the effect of SPF45 overexpression on cell invasion through matrigel in a transwell chamber. In SKOV-3 cells, SPF45 and SPF45-8D significantly increased cell invasion by 1.9-fold and 2.1-fold, respectively, compared with vector control cells, whereas 8A-SPF45 inhibited cell invasion by 45% (Figure 10A and C). In OV2008 cells, SPF45 and SPF45-8D enhanced invasion by 3.2-fold and 3.5-fold, respectively, whereas SPF45-8A did not significantly affect invasion (Figure 10B and D). Collectively, these results show that SPF45 overexpression promoted cell migration and invasion in a phosphorylation-dependent manner.Figure 10.

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