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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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Clk1 enhances SPF45-induced exon 6 exclusion from Fas mRNA. (A) SKOV3 cells were transfected with siRNA against SPF45 (siSPF45) or scrambled control siRNA (scr) for 72 h, and RNA was isolated. Endogenous Fas spliced isoforms were analysed by RT-PCR using primers flanking exon 6. PCR products representing mRNA including exon 6 [Fas (L)] and excluding exon 6 [Fas (S)] are shown and are quantified in the graph, with the ratio Fas (S) to Fas (L) set to one in the control siRNA group. The resuslts are from three independent experiments performed in duplicate and were statistically significant. (B) Parallel cultures to those in (A) were lysed for western blotting using antibodies to SPF45 and actin. (C) Cell lysates from the indicated cell lines were immunoblotted for SPF45 using a polyclonal antibody to recombinant His-SPF45. COS-1 cells transfected with untagged SPF45 (COS-1, +con) and OV2008 cells expressing control or SPF45-specific shRNA served as positive controls. The SPF45 band (SPF45) and an SPF45 degradation product (degr.) in the positive control lane are labelled. (D) COS-1 cells were cotransfected with Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) and endogenous Fas spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratio of exon 6 exclusion from three experiments done in duplicate are shown under the gel images. (E) Schematic of the ΔFas minigene splicing reporter used in transfection assays and the different mRNA isoforms derived from it, representing inclusion or exclusion of exon 6. (F) SPF45 expression induced exon 6 exclusion in a dose-dependent manner. COS-1 cells were co-transfected with ΔFas and increasing amounts of Myc-SPF45. Total RNA was extracted at 24 h and analysed by RT-PCR using ΔFas-specific primers. A representaive gel from at least three independent experiments is shown, and the ratio of the lower band to the upper band is shown below the gel. The lower panel represents a western blot of Myc-SPF45 and actin protein expression from one experiment. (G) Clk1 overexpression promotes SPF45 alternative splicing activity. COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratios of exon 6 exclusion are shown under the gel images. Results were derived from three independent experiments done in duplicate. Statistical significance (P < 0.01) is indicated in the graph. (H) Clk1 overexpression enhanced SPF45 protein levels. Protein lysates were prepared from cells transfected as in (G) and were subjected to western blotting using antibodies to Myc, Clk1 and actin.
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gkt170-F1: Clk1 enhances SPF45-induced exon 6 exclusion from Fas mRNA. (A) SKOV3 cells were transfected with siRNA against SPF45 (siSPF45) or scrambled control siRNA (scr) for 72 h, and RNA was isolated. Endogenous Fas spliced isoforms were analysed by RT-PCR using primers flanking exon 6. PCR products representing mRNA including exon 6 [Fas (L)] and excluding exon 6 [Fas (S)] are shown and are quantified in the graph, with the ratio Fas (S) to Fas (L) set to one in the control siRNA group. The resuslts are from three independent experiments performed in duplicate and were statistically significant. (B) Parallel cultures to those in (A) were lysed for western blotting using antibodies to SPF45 and actin. (C) Cell lysates from the indicated cell lines were immunoblotted for SPF45 using a polyclonal antibody to recombinant His-SPF45. COS-1 cells transfected with untagged SPF45 (COS-1, +con) and OV2008 cells expressing control or SPF45-specific shRNA served as positive controls. The SPF45 band (SPF45) and an SPF45 degradation product (degr.) in the positive control lane are labelled. (D) COS-1 cells were cotransfected with Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) and endogenous Fas spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratio of exon 6 exclusion from three experiments done in duplicate are shown under the gel images. (E) Schematic of the ΔFas minigene splicing reporter used in transfection assays and the different mRNA isoforms derived from it, representing inclusion or exclusion of exon 6. (F) SPF45 expression induced exon 6 exclusion in a dose-dependent manner. COS-1 cells were co-transfected with ΔFas and increasing amounts of Myc-SPF45. Total RNA was extracted at 24 h and analysed by RT-PCR using ΔFas-specific primers. A representaive gel from at least three independent experiments is shown, and the ratio of the lower band to the upper band is shown below the gel. The lower panel represents a western blot of Myc-SPF45 and actin protein expression from one experiment. (G) Clk1 overexpression promotes SPF45 alternative splicing activity. COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratios of exon 6 exclusion are shown under the gel images. Results were derived from three independent experiments done in duplicate. Statistical significance (P < 0.01) is indicated in the graph. (H) Clk1 overexpression enhanced SPF45 protein levels. Protein lysates were prepared from cells transfected as in (G) and were subjected to western blotting using antibodies to Myc, Clk1 and actin.

Mentions: SPF45 enhances exon 6 exclusion from Fas pre-mRNA in a cellular minigene assay (21,33). To determine whether endogenous SPF45 has a similar effect on splicing of endogenous Fas pre-mRNA, we transfected SKOV-3 ovarian cancer cells with siRNA against SPF45. Seventy-two hour after transfection, RNA was harvested, and the endogenous Fas mRNA isoforms containing or excluding exon 6 were analysed by RT-PCR using primers flanking exon 6. SPF45 knockdown inhibited the splicing of the short Fas mRNA isoform (lacking exon 6) from Fas mRNA (Figure 1A). No other bands were detected on the gel. Immunoblotting of parallel cultures with an anti-SPF45 polyclonal antibody confirmed SPF45 knockdown (Figure 1B). This polyclonal antibody generated to recombinant SPF45 protein recognizes endogenous SPF45 from several ovarian cancer cell lines and HeLa cells, with specificity determined by comparison with lysate from COS-1 cells transfected with untagged SPF45 (COS-1 +con) and SPF45 shRNA knockdown in OV2008 cells (Figure 1C). Consistent with the above-splicing results, transient transfection of SPF45 into COS-1 cells increased splicing of the short isoform of endogenous Fas mRNA, demonstrating enhanced exon 6 exclusion (Figure 1D). Clk1 is an SR protein kinase that phosphorylates splicing factors to regulate their localization and splice site utilization (19,20,22–24). To determine whether Clk1-regulated SPF45-dependent exon 6 exclusion from endogenous Fas mRNA, COS-1 cells were cotransfected with Clk1 and SPF45. Overexpression of Clk1 with SPF45 significantly increased exon 6 exclusion from endogenous Fas mRNA compared with transfection of SPF45 and empty vector (Figure 1D). We used our previously described ΔFas minigene (14,21), comprised of exon 5 through exon 7 of genomic Fas, including introns (Figure 1E), to study the effect of Clk1 on SPF45 splice site utilization. Cotransfection of COS-1 cells with ΔFas and increasing amounts of Myc-SPF45 plasmid caused a dose-dependent increase in exon 6 exclusion, shown as an increase in the lower band, demonstrating that our ΔFas minigene worked as a target of SPF45 in cells (Figure 1F) (21). COS-1 cells were chosen for minigene splicing assays owing to their low expression of endogenous SPF45 (Figure 1C), which allowed us to better determine SPF45-specific effects on alternative splicing of ΔFas mRNA. Clk1 was co-transfected into cells with SPF45 and the ΔFas minigene and exon 6 exclusion of ΔFas was determined. Clk1 enhanced SPF45-induced exon 6 excluion of ΔFas by >50% (Figure 1G), suggesting that SPF45 may be a cellular target of Clk1. Interestingly, Clk1 overexpression also enhanced Myc-SPF45 protein expression (Figure 1H). As SPF45 concentration directly affects splice site utilization (Figure 1F), the enhancement of SPF45 expression by Clk1 could be a possible mechanism for the enhanced exon 6 exclusion and was investigated futher.Figure 1.


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)

Clk1 enhances SPF45-induced exon 6 exclusion from Fas mRNA. (A) SKOV3 cells were transfected with siRNA against SPF45 (siSPF45) or scrambled control siRNA (scr) for 72 h, and RNA was isolated. Endogenous Fas spliced isoforms were analysed by RT-PCR using primers flanking exon 6. PCR products representing mRNA including exon 6 [Fas (L)] and excluding exon 6 [Fas (S)] are shown and are quantified in the graph, with the ratio Fas (S) to Fas (L) set to one in the control siRNA group. The resuslts are from three independent experiments performed in duplicate and were statistically significant. (B) Parallel cultures to those in (A) were lysed for western blotting using antibodies to SPF45 and actin. (C) Cell lysates from the indicated cell lines were immunoblotted for SPF45 using a polyclonal antibody to recombinant His-SPF45. COS-1 cells transfected with untagged SPF45 (COS-1, +con) and OV2008 cells expressing control or SPF45-specific shRNA served as positive controls. The SPF45 band (SPF45) and an SPF45 degradation product (degr.) in the positive control lane are labelled. (D) COS-1 cells were cotransfected with Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) and endogenous Fas spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratio of exon 6 exclusion from three experiments done in duplicate are shown under the gel images. (E) Schematic of the ΔFas minigene splicing reporter used in transfection assays and the different mRNA isoforms derived from it, representing inclusion or exclusion of exon 6. (F) SPF45 expression induced exon 6 exclusion in a dose-dependent manner. COS-1 cells were co-transfected with ΔFas and increasing amounts of Myc-SPF45. Total RNA was extracted at 24 h and analysed by RT-PCR using ΔFas-specific primers. A representaive gel from at least three independent experiments is shown, and the ratio of the lower band to the upper band is shown below the gel. The lower panel represents a western blot of Myc-SPF45 and actin protein expression from one experiment. (G) Clk1 overexpression promotes SPF45 alternative splicing activity. COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratios of exon 6 exclusion are shown under the gel images. Results were derived from three independent experiments done in duplicate. Statistical significance (P < 0.01) is indicated in the graph. (H) Clk1 overexpression enhanced SPF45 protein levels. Protein lysates were prepared from cells transfected as in (G) and were subjected to western blotting using antibodies to Myc, Clk1 and actin.
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gkt170-F1: Clk1 enhances SPF45-induced exon 6 exclusion from Fas mRNA. (A) SKOV3 cells were transfected with siRNA against SPF45 (siSPF45) or scrambled control siRNA (scr) for 72 h, and RNA was isolated. Endogenous Fas spliced isoforms were analysed by RT-PCR using primers flanking exon 6. PCR products representing mRNA including exon 6 [Fas (L)] and excluding exon 6 [Fas (S)] are shown and are quantified in the graph, with the ratio Fas (S) to Fas (L) set to one in the control siRNA group. The resuslts are from three independent experiments performed in duplicate and were statistically significant. (B) Parallel cultures to those in (A) were lysed for western blotting using antibodies to SPF45 and actin. (C) Cell lysates from the indicated cell lines were immunoblotted for SPF45 using a polyclonal antibody to recombinant His-SPF45. COS-1 cells transfected with untagged SPF45 (COS-1, +con) and OV2008 cells expressing control or SPF45-specific shRNA served as positive controls. The SPF45 band (SPF45) and an SPF45 degradation product (degr.) in the positive control lane are labelled. (D) COS-1 cells were cotransfected with Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) and endogenous Fas spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratio of exon 6 exclusion from three experiments done in duplicate are shown under the gel images. (E) Schematic of the ΔFas minigene splicing reporter used in transfection assays and the different mRNA isoforms derived from it, representing inclusion or exclusion of exon 6. (F) SPF45 expression induced exon 6 exclusion in a dose-dependent manner. COS-1 cells were co-transfected with ΔFas and increasing amounts of Myc-SPF45. Total RNA was extracted at 24 h and analysed by RT-PCR using ΔFas-specific primers. A representaive gel from at least three independent experiments is shown, and the ratio of the lower band to the upper band is shown below the gel. The lower panel represents a western blot of Myc-SPF45 and actin protein expression from one experiment. (G) Clk1 overexpression promotes SPF45 alternative splicing activity. COS-1 cells were cotransfected with ΔFas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratios of exon 6 exclusion are shown under the gel images. Results were derived from three independent experiments done in duplicate. Statistical significance (P < 0.01) is indicated in the graph. (H) Clk1 overexpression enhanced SPF45 protein levels. Protein lysates were prepared from cells transfected as in (G) and were subjected to western blotting using antibodies to Myc, Clk1 and actin.
Mentions: SPF45 enhances exon 6 exclusion from Fas pre-mRNA in a cellular minigene assay (21,33). To determine whether endogenous SPF45 has a similar effect on splicing of endogenous Fas pre-mRNA, we transfected SKOV-3 ovarian cancer cells with siRNA against SPF45. Seventy-two hour after transfection, RNA was harvested, and the endogenous Fas mRNA isoforms containing or excluding exon 6 were analysed by RT-PCR using primers flanking exon 6. SPF45 knockdown inhibited the splicing of the short Fas mRNA isoform (lacking exon 6) from Fas mRNA (Figure 1A). No other bands were detected on the gel. Immunoblotting of parallel cultures with an anti-SPF45 polyclonal antibody confirmed SPF45 knockdown (Figure 1B). This polyclonal antibody generated to recombinant SPF45 protein recognizes endogenous SPF45 from several ovarian cancer cell lines and HeLa cells, with specificity determined by comparison with lysate from COS-1 cells transfected with untagged SPF45 (COS-1 +con) and SPF45 shRNA knockdown in OV2008 cells (Figure 1C). Consistent with the above-splicing results, transient transfection of SPF45 into COS-1 cells increased splicing of the short isoform of endogenous Fas mRNA, demonstrating enhanced exon 6 exclusion (Figure 1D). Clk1 is an SR protein kinase that phosphorylates splicing factors to regulate their localization and splice site utilization (19,20,22–24). To determine whether Clk1-regulated SPF45-dependent exon 6 exclusion from endogenous Fas mRNA, COS-1 cells were cotransfected with Clk1 and SPF45. Overexpression of Clk1 with SPF45 significantly increased exon 6 exclusion from endogenous Fas mRNA compared with transfection of SPF45 and empty vector (Figure 1D). We used our previously described ΔFas minigene (14,21), comprised of exon 5 through exon 7 of genomic Fas, including introns (Figure 1E), to study the effect of Clk1 on SPF45 splice site utilization. Cotransfection of COS-1 cells with ΔFas and increasing amounts of Myc-SPF45 plasmid caused a dose-dependent increase in exon 6 exclusion, shown as an increase in the lower band, demonstrating that our ΔFas minigene worked as a target of SPF45 in cells (Figure 1F) (21). COS-1 cells were chosen for minigene splicing assays owing to their low expression of endogenous SPF45 (Figure 1C), which allowed us to better determine SPF45-specific effects on alternative splicing of ΔFas mRNA. Clk1 was co-transfected into cells with SPF45 and the ΔFas minigene and exon 6 exclusion of ΔFas was determined. Clk1 enhanced SPF45-induced exon 6 excluion of ΔFas by >50% (Figure 1G), suggesting that SPF45 may be a cellular target of Clk1. Interestingly, Clk1 overexpression also enhanced Myc-SPF45 protein expression (Figure 1H). As SPF45 concentration directly affects splice site utilization (Figure 1F), the enhancement of SPF45 expression by Clk1 could be a possible mechanism for the enhanced exon 6 exclusion and was investigated futher.Figure 1.

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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Related in: MedlinePlus