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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 inhibits alternative splicing of cortactin and enhanced phosphorylation of cortactin and ERK in a phosphorylation site-dependent manner. (A) Schematic of cortactin alternative splicing, showing exon inclusion in wt cortactin and the two known SVs, representing inclusion or exclusion of exons 10 and 11 (48). Arrows indicate the position of primers to detect total cortactin and wild-type (wt) cortactin. (B) SPF45 and SPF45-8D enhance splicing of wild-type cortactin. Quantitative real-time PCR analysis of wild-type cortactin and total cortactin (wt + SV1 + SV2) from SKOV-3 cell lines stably overexpressing wt-SPF45, SPF45-8A, SPF45-8D or empty vector. The results, expressed as the ratio of wt-cortactin to total cortactin, are from three experiments done in duplicate and are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt-SPF45 group. (C) SPF45 and SPF45-8D overexpression enhance ERK activation and cortactin phosphorylation. Protein lysates from SKOV-3 stable cell lines were immunoblotted for phosphorylated and total ERK and cortactin. S and L signify short and long exposure. (D) The ratio of wild-type to total cortactin was measured in OV2008 stable cell lines as in (B). (E) Total and phosphorylated ERK and cortactin protein were measured by immunoblotting OV2008 cell lysates as in (C). All immunoblots are representative images from three to four independent experiments.
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gkt170-F12: SPF45 overexpression inhibits alternative splicing of cortactin and enhanced phosphorylation of cortactin and ERK in a phosphorylation site-dependent manner. (A) Schematic of cortactin alternative splicing, showing exon inclusion in wt cortactin and the two known SVs, representing inclusion or exclusion of exons 10 and 11 (48). Arrows indicate the position of primers to detect total cortactin and wild-type (wt) cortactin. (B) SPF45 and SPF45-8D enhance splicing of wild-type cortactin. Quantitative real-time PCR analysis of wild-type cortactin and total cortactin (wt + SV1 + SV2) from SKOV-3 cell lines stably overexpressing wt-SPF45, SPF45-8A, SPF45-8D or empty vector. The results, expressed as the ratio of wt-cortactin to total cortactin, are from three experiments done in duplicate and are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt-SPF45 group. (C) SPF45 and SPF45-8D overexpression enhance ERK activation and cortactin phosphorylation. Protein lysates from SKOV-3 stable cell lines were immunoblotted for phosphorylated and total ERK and cortactin. S and L signify short and long exposure. (D) The ratio of wild-type to total cortactin was measured in OV2008 stable cell lines as in (B). (E) Total and phosphorylated ERK and cortactin protein were measured by immunoblotting OV2008 cell lysates as in (C). All immunoblots are representative images from three to four independent experiments.

Mentions: Analysis of alternative splicing in SKOV-3-Myc-SPF45 cells identified the filamentous actin-binding protein cortactin as a potential splicing target of SPF45 (Al-Ayoubi and Eblen, unpublished data). Cortactin overexpression enhances cell migration and invasion (47), whereas cortactin splice variant (SV) 1 induces less migration and SV2 inhibits migration when overexpressed (48). The qRT-PCR using primers specific to wt-cortactin or total cortactin (wt + SV1 + SV2) (Figure 12A) in the SKOV-3 stable cell lines showed that SPF45 and SPF45-8D significantly increased the ratio of wt:total cortactin compared with vector control cells by 64 and 72%, respectively, whereas SPF45-8A did not show a significant increase (Figure 12B). These differences were also evident at the protein level (Figure 12C), where the wt-cortactin doublet and faster migrating SV1, but not SV2, were observed. In addition, expression of SPF45 and SPF45-8D enhanced ERK activation and phosphorylation of Ser405 and Ser418 on cortactin (Figure 12C), two ERK-dependent sites that enhance cortactin function (39,49,50). Similar results were obtained in OV2008 stable cell lines (Figure 12D and E).Figure 12.


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 inhibits alternative splicing of cortactin and enhanced phosphorylation of cortactin and ERK in a phosphorylation site-dependent manner. (A) Schematic of cortactin alternative splicing, showing exon inclusion in wt cortactin and the two known SVs, representing inclusion or exclusion of exons 10 and 11 (48). Arrows indicate the position of primers to detect total cortactin and wild-type (wt) cortactin. (B) SPF45 and SPF45-8D enhance splicing of wild-type cortactin. Quantitative real-time PCR analysis of wild-type cortactin and total cortactin (wt + SV1 + SV2) from SKOV-3 cell lines stably overexpressing wt-SPF45, SPF45-8A, SPF45-8D or empty vector. The results, expressed as the ratio of wt-cortactin to total cortactin, are from three experiments done in duplicate and are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt-SPF45 group. (C) SPF45 and SPF45-8D overexpression enhance ERK activation and cortactin phosphorylation. Protein lysates from SKOV-3 stable cell lines were immunoblotted for phosphorylated and total ERK and cortactin. S and L signify short and long exposure. (D) The ratio of wild-type to total cortactin was measured in OV2008 stable cell lines as in (B). (E) Total and phosphorylated ERK and cortactin protein were measured by immunoblotting OV2008 cell lysates as in (C). All immunoblots are representative images from three to four independent experiments.
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gkt170-F12: SPF45 overexpression inhibits alternative splicing of cortactin and enhanced phosphorylation of cortactin and ERK in a phosphorylation site-dependent manner. (A) Schematic of cortactin alternative splicing, showing exon inclusion in wt cortactin and the two known SVs, representing inclusion or exclusion of exons 10 and 11 (48). Arrows indicate the position of primers to detect total cortactin and wild-type (wt) cortactin. (B) SPF45 and SPF45-8D enhance splicing of wild-type cortactin. Quantitative real-time PCR analysis of wild-type cortactin and total cortactin (wt + SV1 + SV2) from SKOV-3 cell lines stably overexpressing wt-SPF45, SPF45-8A, SPF45-8D or empty vector. The results, expressed as the ratio of wt-cortactin to total cortactin, are from three experiments done in duplicate and are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt-SPF45 group. (C) SPF45 and SPF45-8D overexpression enhance ERK activation and cortactin phosphorylation. Protein lysates from SKOV-3 stable cell lines were immunoblotted for phosphorylated and total ERK and cortactin. S and L signify short and long exposure. (D) The ratio of wild-type to total cortactin was measured in OV2008 stable cell lines as in (B). (E) Total and phosphorylated ERK and cortactin protein were measured by immunoblotting OV2008 cell lysates as in (C). All immunoblots are representative images from three to four independent experiments.
Mentions: Analysis of alternative splicing in SKOV-3-Myc-SPF45 cells identified the filamentous actin-binding protein cortactin as a potential splicing target of SPF45 (Al-Ayoubi and Eblen, unpublished data). Cortactin overexpression enhances cell migration and invasion (47), whereas cortactin splice variant (SV) 1 induces less migration and SV2 inhibits migration when overexpressed (48). The qRT-PCR using primers specific to wt-cortactin or total cortactin (wt + SV1 + SV2) (Figure 12A) in the SKOV-3 stable cell lines showed that SPF45 and SPF45-8D significantly increased the ratio of wt:total cortactin compared with vector control cells by 64 and 72%, respectively, whereas SPF45-8A did not show a significant increase (Figure 12B). These differences were also evident at the protein level (Figure 12C), where the wt-cortactin doublet and faster migrating SV1, but not SV2, were observed. In addition, expression of SPF45 and SPF45-8D enhanced ERK activation and phosphorylation of Ser405 and Ser418 on cortactin (Figure 12C), two ERK-dependent sites that enhance cortactin function (39,49,50). Similar results were obtained in OV2008 stable cell lines (Figure 12D and E).Figure 12.

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