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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 inhibition decreases the half-life of SPF45 through a proteasome-dependent pathway. (A) A2780 cells were treated with cycloheximide (50 μg/ml) and TG003 (10 μM) or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies to detect endogenous proteins. (B) Graph of SPF45 protein levels relative to the actin loading control from (A). (C) SKOV-3-Myc-SPF45 stable cells were treated with cycloheximide and TG003 or DMSO for the indicated times followed by western blot analysis with anti-Myc and anti-actin antibodies. (D) Graph of Myc-SPF45 protein levels relative to actin from (C). (E) Knockdown of Clk1 inhibits expression of endogenous SPF45. SKOV-3 cells and HeLa cells were transfected with siRNA to Clk1 or control siRNA (scr) for 48 h. Cell lysates were immunoblotted for endogenous Clk1, SPF45 and actin. (F) The increased degradation of SPF45 with Clk1 inhibition is proteasome-dependent. A2780 cells were treated with MG132 (10 μM) and TG003 or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies. Quantitation of SFP45 expression relative to actin is below each lane, with the DMSO control lane set to 1.
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gkt170-F4: Clk1 inhibition decreases the half-life of SPF45 through a proteasome-dependent pathway. (A) A2780 cells were treated with cycloheximide (50 μg/ml) and TG003 (10 μM) or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies to detect endogenous proteins. (B) Graph of SPF45 protein levels relative to the actin loading control from (A). (C) SKOV-3-Myc-SPF45 stable cells were treated with cycloheximide and TG003 or DMSO for the indicated times followed by western blot analysis with anti-Myc and anti-actin antibodies. (D) Graph of Myc-SPF45 protein levels relative to actin from (C). (E) Knockdown of Clk1 inhibits expression of endogenous SPF45. SKOV-3 cells and HeLa cells were transfected with siRNA to Clk1 or control siRNA (scr) for 48 h. Cell lysates were immunoblotted for endogenous Clk1, SPF45 and actin. (F) The increased degradation of SPF45 with Clk1 inhibition is proteasome-dependent. A2780 cells were treated with MG132 (10 μM) and TG003 or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies. Quantitation of SFP45 expression relative to actin is below each lane, with the DMSO control lane set to 1.

Mentions: We suspected that the enhancement or inhibition of exon 6 exclusion by SPF45 with Clk1 activation or inhibition, respectively, was partially due to regulation of SPF45 protein levels, as SPF45 expression directly impacts exon 6 exclusion (Figure 1F). To determine whether Clk1 regulated the half-life of SPF45, A2780 ovarian cancer cells, which express high levels of endogenous SPF45 (Figure 1C), were treated with cycloheximide in the absence or presence of TG003 for 24 h (Figure 4A). TG003 significantly decreased the half-life of endogenous SPF45 from 15 to 8 h (Figure 4B). In addition, SKOV-3 ovarian cancer cells stably expressing Myc-SPF45 were treated with DMSO or TG003 in the presence of cycloheximide. Consistent with the aforementioned results, TG003 decreased the half-life of Myc-SPF45 from 16 to 7 h (Figure 4C and D). Moreover, knockdown of endogenous Clk1 with siRNA decreased endogenous SPF45 protein levels in both SKOV-3 and HeLa cells (Figure 4E). To determine whether the decrease in SPF45 half-life in the presence of TG003 was dependent on the proteasome, A2780 cells were treated with the proteasome inhibitor MG132 in the absence or presence of TG003 (Figure 4F). MG132 co-treatment with TG003 prevented the downregulation of SPF45 protein levels that was induced by TG003 treatment, indicating that Clk1 inhibition regulated SPF45 degradation through a proteasome-dependent pathway.Figure 4.


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 inhibition decreases the half-life of SPF45 through a proteasome-dependent pathway. (A) A2780 cells were treated with cycloheximide (50 μg/ml) and TG003 (10 μM) or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies to detect endogenous proteins. (B) Graph of SPF45 protein levels relative to the actin loading control from (A). (C) SKOV-3-Myc-SPF45 stable cells were treated with cycloheximide and TG003 or DMSO for the indicated times followed by western blot analysis with anti-Myc and anti-actin antibodies. (D) Graph of Myc-SPF45 protein levels relative to actin from (C). (E) Knockdown of Clk1 inhibits expression of endogenous SPF45. SKOV-3 cells and HeLa cells were transfected with siRNA to Clk1 or control siRNA (scr) for 48 h. Cell lysates were immunoblotted for endogenous Clk1, SPF45 and actin. (F) The increased degradation of SPF45 with Clk1 inhibition is proteasome-dependent. A2780 cells were treated with MG132 (10 μM) and TG003 or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies. Quantitation of SFP45 expression relative to actin is below each lane, with the DMSO control lane set to 1.
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gkt170-F4: Clk1 inhibition decreases the half-life of SPF45 through a proteasome-dependent pathway. (A) A2780 cells were treated with cycloheximide (50 μg/ml) and TG003 (10 μM) or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies to detect endogenous proteins. (B) Graph of SPF45 protein levels relative to the actin loading control from (A). (C) SKOV-3-Myc-SPF45 stable cells were treated with cycloheximide and TG003 or DMSO for the indicated times followed by western blot analysis with anti-Myc and anti-actin antibodies. (D) Graph of Myc-SPF45 protein levels relative to actin from (C). (E) Knockdown of Clk1 inhibits expression of endogenous SPF45. SKOV-3 cells and HeLa cells were transfected with siRNA to Clk1 or control siRNA (scr) for 48 h. Cell lysates were immunoblotted for endogenous Clk1, SPF45 and actin. (F) The increased degradation of SPF45 with Clk1 inhibition is proteasome-dependent. A2780 cells were treated with MG132 (10 μM) and TG003 or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies. Quantitation of SFP45 expression relative to actin is below each lane, with the DMSO control lane set to 1.
Mentions: We suspected that the enhancement or inhibition of exon 6 exclusion by SPF45 with Clk1 activation or inhibition, respectively, was partially due to regulation of SPF45 protein levels, as SPF45 expression directly impacts exon 6 exclusion (Figure 1F). To determine whether Clk1 regulated the half-life of SPF45, A2780 ovarian cancer cells, which express high levels of endogenous SPF45 (Figure 1C), were treated with cycloheximide in the absence or presence of TG003 for 24 h (Figure 4A). TG003 significantly decreased the half-life of endogenous SPF45 from 15 to 8 h (Figure 4B). In addition, SKOV-3 ovarian cancer cells stably expressing Myc-SPF45 were treated with DMSO or TG003 in the presence of cycloheximide. Consistent with the aforementioned results, TG003 decreased the half-life of Myc-SPF45 from 16 to 7 h (Figure 4C and D). Moreover, knockdown of endogenous Clk1 with siRNA decreased endogenous SPF45 protein levels in both SKOV-3 and HeLa cells (Figure 4E). To determine whether the decrease in SPF45 half-life in the presence of TG003 was dependent on the proteasome, A2780 cells were treated with the proteasome inhibitor MG132 in the absence or presence of TG003 (Figure 4F). MG132 co-treatment with TG003 prevented the downregulation of SPF45 protein levels that was induced by TG003 treatment, indicating that Clk1 inhibition regulated SPF45 degradation through a proteasome-dependent pathway.Figure 4.

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