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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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Mutation of Clk1 phosphorylation sites on SPF45 regulates SPF45 splice site utilization. (A) COS-1 cells were transfected for ΔFas splicing assays as above using either Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D in the absence or presence of either empty vector or Clk1. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. (B) The means and SE for the relative ratios of exon 6 exclusion from (A) are shown in the graph. Results were derived from three independent experiments in duplicate and statistical significance (P < 0.01) is indicated. (C) The same as in (A), but total RNA was subjected to RT-PCR analysis using primers specific to Myc-SPF45 and GAPDH. (D) Protein lysates from cells transfected in parallel were immunoblotted with anti-Myc and anti-actin antibodies.
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gkt170-F6: Mutation of Clk1 phosphorylation sites on SPF45 regulates SPF45 splice site utilization. (A) COS-1 cells were transfected for ΔFas splicing assays as above using either Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D in the absence or presence of either empty vector or Clk1. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. (B) The means and SE for the relative ratios of exon 6 exclusion from (A) are shown in the graph. Results were derived from three independent experiments in duplicate and statistical significance (P < 0.01) is indicated. (C) The same as in (A), but total RNA was subjected to RT-PCR analysis using primers specific to Myc-SPF45 and GAPDH. (D) Protein lysates from cells transfected in parallel were immunoblotted with anti-Myc and anti-actin antibodies.

Mentions: To determine the collective effect of Clk1 phosphorylation on SPF45, we compared exon 6 exclusion by SPF45, SPF45-8A or a phospho-mimetic SPF45-8D in the ΔFas minigene assay. Surprisingly, Myc-SPF45-8A demonstrated a significant 25% increase in exon 6 exclusion compared with wild-type SPF45 in the absence of exogenous Clk1, suggesting that phosphorylation of these sites in total inhibited SPF45-induced exon 6 exclusion (Figure 6A and B). Similarly, mutation of these eight residues to aspartate inhibited exon 6 exclusion by a comparable amount. When Clk1 was co-expressed, wild-type Myc-SPF45 induction of exon 6 was enhanced, as aforementioned, but the significant difference between wild-type Myc-SPF45 and Myc-SPF45-8A was lost, suggesting that Clk1 had little effect on SPF45 exon 6 exclusion when the 8 phosphorylation sites were mutated to alanine. Similarly, Clk1 expression had little effect on Myc-SPF45-8D-induced exon 6 exclusion. SPF45 mRNA expression was unaffected by Clk1 expression (Figure 6C), whereas expression of the mutant proteins was similar to wild-type (Figure 6D).Figure 6.


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)

Mutation of Clk1 phosphorylation sites on SPF45 regulates SPF45 splice site utilization. (A) COS-1 cells were transfected for ΔFas splicing assays as above using either Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D in the absence or presence of either empty vector or Clk1. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. (B) The means and SE for the relative ratios of exon 6 exclusion from (A) are shown in the graph. Results were derived from three independent experiments in duplicate and statistical significance (P < 0.01) is indicated. (C) The same as in (A), but total RNA was subjected to RT-PCR analysis using primers specific to Myc-SPF45 and GAPDH. (D) Protein lysates from cells transfected in parallel were immunoblotted with anti-Myc and anti-actin antibodies.
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Related In: Results  -  Collection

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gkt170-F6: Mutation of Clk1 phosphorylation sites on SPF45 regulates SPF45 splice site utilization. (A) COS-1 cells were transfected for ΔFas splicing assays as above using either Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D in the absence or presence of either empty vector or Clk1. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. (B) The means and SE for the relative ratios of exon 6 exclusion from (A) are shown in the graph. Results were derived from three independent experiments in duplicate and statistical significance (P < 0.01) is indicated. (C) The same as in (A), but total RNA was subjected to RT-PCR analysis using primers specific to Myc-SPF45 and GAPDH. (D) Protein lysates from cells transfected in parallel were immunoblotted with anti-Myc and anti-actin antibodies.
Mentions: To determine the collective effect of Clk1 phosphorylation on SPF45, we compared exon 6 exclusion by SPF45, SPF45-8A or a phospho-mimetic SPF45-8D in the ΔFas minigene assay. Surprisingly, Myc-SPF45-8A demonstrated a significant 25% increase in exon 6 exclusion compared with wild-type SPF45 in the absence of exogenous Clk1, suggesting that phosphorylation of these sites in total inhibited SPF45-induced exon 6 exclusion (Figure 6A and B). Similarly, mutation of these eight residues to aspartate inhibited exon 6 exclusion by a comparable amount. When Clk1 was co-expressed, wild-type Myc-SPF45 induction of exon 6 was enhanced, as aforementioned, but the significant difference between wild-type Myc-SPF45 and Myc-SPF45-8A was lost, suggesting that Clk1 had little effect on SPF45 exon 6 exclusion when the 8 phosphorylation sites were mutated to alanine. Similarly, Clk1 expression had little effect on Myc-SPF45-8D-induced exon 6 exclusion. SPF45 mRNA expression was unaffected by Clk1 expression (Figure 6C), whereas expression of the mutant proteins was similar to wild-type (Figure 6D).Figure 6.

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