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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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Inhibition of Clk1 decreases both SPF45 protein levels and exon 6 exclusion by SPF45. (A) COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) or Clk1-K233R (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The graph under the gel image represents the quantification of the corresponding bands from three experiments done in duplicate. (B) COS-1 cells were cotransfected as in (A), and whole-cell protein lysates were immunoblotted with anti-Myc and anti-actin antibodies. (C) COS-1 cells were pretreated with the Clk inhibitor TG003 (10 μM) for 1 h and then cotransfected with ΔFas and Myc-SPF45 or empty vector. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. A graph under the gel image showed the relative ratio of the lower band to the upper band. The results were derived from three independent experiments done in duplicate and statistical significance (P < 0.01) is indicated. (D) COS-1 cells were pretreated with TG003 for 1 h and then cotransfected as in (C). Whole-cell protein lysates were immunoblotted as in (B).
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gkt170-F2: Inhibition of Clk1 decreases both SPF45 protein levels and exon 6 exclusion by SPF45. (A) COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) or Clk1-K233R (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The graph under the gel image represents the quantification of the corresponding bands from three experiments done in duplicate. (B) COS-1 cells were cotransfected as in (A), and whole-cell protein lysates were immunoblotted with anti-Myc and anti-actin antibodies. (C) COS-1 cells were pretreated with the Clk inhibitor TG003 (10 μM) for 1 h and then cotransfected with ΔFas and Myc-SPF45 or empty vector. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. A graph under the gel image showed the relative ratio of the lower band to the upper band. The results were derived from three independent experiments done in duplicate and statistical significance (P < 0.01) is indicated. (D) COS-1 cells were pretreated with TG003 for 1 h and then cotransfected as in (C). Whole-cell protein lysates were immunoblotted as in (B).

Mentions: To further determine the role of Clk1 kinase activity in regulating SPF45 exon 6 exclusion of Fas pre-mRNA, we generated a kinase-dead Clk1, Clk1-K233R, and co-transfected COS-1 cells with plasmids for SPF45 and ΔFas. Clk1-K233R decreased both SPF45-induced exon 6 exclusion (Figure 2A) and SPF45 protein levels (Figure 2B) compared with wild-type Clk1. To confirm this result, a selective Clk1 inhibitor, TG003 (41), was used to block endogenous Clk1 activity. TG003 not only inhibited the increase of exon 6 exclusion induced by SPF45 (Figure 2C) but also decreased SPF45 protein expression (Figure 2D), similar to what was seen with kinase-dead Clk1 expression. Next, we used siRNA to knock down endogenous Clk1 expression. Transfection of cells with any of three different siRNA-targeting Clk1 led to a signifcant decrease in endogenous Clk1 mRNA and protein levels after 48 h, as determined by real-time PCR and western blot analysis, respectively (Figure 3A and B). The siRNA-mediated knockdown of Clk1 in COS-1 cells significantly decreased exon 6 exclusion in an SPF45-dependent manner (Figure 3C and D) and greatly inhibited Myc-SPF45 protein expression (Figure 3E), most likely contributing to the reduction in exon 6 exclusion.Figure 2.


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)

Inhibition of Clk1 decreases both SPF45 protein levels and exon 6 exclusion by SPF45. (A) COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) or Clk1-K233R (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The graph under the gel image represents the quantification of the corresponding bands from three experiments done in duplicate. (B) COS-1 cells were cotransfected as in (A), and whole-cell protein lysates were immunoblotted with anti-Myc and anti-actin antibodies. (C) COS-1 cells were pretreated with the Clk inhibitor TG003 (10 μM) for 1 h and then cotransfected with ΔFas and Myc-SPF45 or empty vector. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. A graph under the gel image showed the relative ratio of the lower band to the upper band. The results were derived from three independent experiments done in duplicate and statistical significance (P < 0.01) is indicated. (D) COS-1 cells were pretreated with TG003 for 1 h and then cotransfected as in (C). Whole-cell protein lysates were immunoblotted as in (B).
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gkt170-F2: Inhibition of Clk1 decreases both SPF45 protein levels and exon 6 exclusion by SPF45. (A) COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) or Clk1-K233R (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The graph under the gel image represents the quantification of the corresponding bands from three experiments done in duplicate. (B) COS-1 cells were cotransfected as in (A), and whole-cell protein lysates were immunoblotted with anti-Myc and anti-actin antibodies. (C) COS-1 cells were pretreated with the Clk inhibitor TG003 (10 μM) for 1 h and then cotransfected with ΔFas and Myc-SPF45 or empty vector. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. A graph under the gel image showed the relative ratio of the lower band to the upper band. The results were derived from three independent experiments done in duplicate and statistical significance (P < 0.01) is indicated. (D) COS-1 cells were pretreated with TG003 for 1 h and then cotransfected as in (C). Whole-cell protein lysates were immunoblotted as in (B).
Mentions: To further determine the role of Clk1 kinase activity in regulating SPF45 exon 6 exclusion of Fas pre-mRNA, we generated a kinase-dead Clk1, Clk1-K233R, and co-transfected COS-1 cells with plasmids for SPF45 and ΔFas. Clk1-K233R decreased both SPF45-induced exon 6 exclusion (Figure 2A) and SPF45 protein levels (Figure 2B) compared with wild-type Clk1. To confirm this result, a selective Clk1 inhibitor, TG003 (41), was used to block endogenous Clk1 activity. TG003 not only inhibited the increase of exon 6 exclusion induced by SPF45 (Figure 2C) but also decreased SPF45 protein expression (Figure 2D), similar to what was seen with kinase-dead Clk1 expression. Next, we used siRNA to knock down endogenous Clk1 expression. Transfection of cells with any of three different siRNA-targeting Clk1 led to a signifcant decrease in endogenous Clk1 mRNA and protein levels after 48 h, as determined by real-time PCR and western blot analysis, respectively (Figure 3A and B). The siRNA-mediated knockdown of Clk1 in COS-1 cells significantly decreased exon 6 exclusion in an SPF45-dependent manner (Figure 3C and D) and greatly inhibited Myc-SPF45 protein expression (Figure 3E), most likely contributing to the reduction in exon 6 exclusion.Figure 2.

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