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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 the Clk1 phosphorylation sites in SPF45 affects ΔFas mRNA binding, but not binding to other splicing factors. (A) COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. After 24 h, the cells were lysed, and anti-Myc immunoprecipitates were run on a gel and immunoblotted for Myc and co-immunoprecipitating endogenous SF3b155, U2AF65 and SF1. Cell lysates (Lys.) were immunoblotted as indicated. (B) COS-1 cells were co-transfected with ΔFas and either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. Twenty-four hour after transfection, the cells were subjected to RNA IP using anti-Myc antibody followed by RT-qPCR analysis to detect the binding of ΔFas mRNA to Myc-SPF45. Relative binding to the control IP from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.
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gkt170-F8: Mutation of the Clk1 phosphorylation sites in SPF45 affects ΔFas mRNA binding, but not binding to other splicing factors. (A) COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. After 24 h, the cells were lysed, and anti-Myc immunoprecipitates were run on a gel and immunoblotted for Myc and co-immunoprecipitating endogenous SF3b155, U2AF65 and SF1. Cell lysates (Lys.) were immunoblotted as indicated. (B) COS-1 cells were co-transfected with ΔFas and either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. Twenty-four hour after transfection, the cells were subjected to RNA IP using anti-Myc antibody followed by RT-qPCR analysis to detect the binding of ΔFas mRNA to Myc-SPF45. Relative binding to the control IP from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.

Mentions: SPF45 has been shown to associate with the splicing factors SF3b155, U2AF65 and SF1 through residues in its RRM domain (33,42). To determine whether SPF45 phosphorylation on the Clk1 sites altered binding to these proteins, COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8A or Myc-SPF45-8D. Anti-Myc immunoprecipitates were immunoblotted for endogenous splicing factors. All three splicing factors bound equally well to Myc-SPF45 and either of the SPF45 mutants (Figure 8A), suggesting that Clk1 phosphorylation of SPF45 does not significantly affect association with these proteins. To determine whether phosphorylation affected SPF45 binding to ΔFas RNA, we performed RIP analysis on COS-1 cells, which were cotransfected with ΔFas and wild-type or mutant Myc-SPF45. The ΔFas mRNA detected in anti-Myc immunoprecipitates was analysed by qRT-PCR and was shown to specifically interact with Myc-SPF45 (Figure 8B). Myc-SPF45-8A showed enhanced binding to ΔFas mRNA, whereas Myc-SPF45-8D showed reduced binding, with both reflecting the relative changes in exon 6 exclusion observed in the minigene splicing assays. These data suggest that SPF45 phosphorylation affects Fas mRNA binding.Figure 8.


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 the Clk1 phosphorylation sites in SPF45 affects ΔFas mRNA binding, but not binding to other splicing factors. (A) COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. After 24 h, the cells were lysed, and anti-Myc immunoprecipitates were run on a gel and immunoblotted for Myc and co-immunoprecipitating endogenous SF3b155, U2AF65 and SF1. Cell lysates (Lys.) were immunoblotted as indicated. (B) COS-1 cells were co-transfected with ΔFas and either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. Twenty-four hour after transfection, the cells were subjected to RNA IP using anti-Myc antibody followed by RT-qPCR analysis to detect the binding of ΔFas mRNA to Myc-SPF45. Relative binding to the control IP from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.
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gkt170-F8: Mutation of the Clk1 phosphorylation sites in SPF45 affects ΔFas mRNA binding, but not binding to other splicing factors. (A) COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. After 24 h, the cells were lysed, and anti-Myc immunoprecipitates were run on a gel and immunoblotted for Myc and co-immunoprecipitating endogenous SF3b155, U2AF65 and SF1. Cell lysates (Lys.) were immunoblotted as indicated. (B) COS-1 cells were co-transfected with ΔFas and either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. Twenty-four hour after transfection, the cells were subjected to RNA IP using anti-Myc antibody followed by RT-qPCR analysis to detect the binding of ΔFas mRNA to Myc-SPF45. Relative binding to the control IP from six experiments are shown as mean ± SE. *P < 0.05 versus vector group, #P < 0.05 versus wt-SPF45 group.
Mentions: SPF45 has been shown to associate with the splicing factors SF3b155, U2AF65 and SF1 through residues in its RRM domain (33,42). To determine whether SPF45 phosphorylation on the Clk1 sites altered binding to these proteins, COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8A or Myc-SPF45-8D. Anti-Myc immunoprecipitates were immunoblotted for endogenous splicing factors. All three splicing factors bound equally well to Myc-SPF45 and either of the SPF45 mutants (Figure 8A), suggesting that Clk1 phosphorylation of SPF45 does not significantly affect association with these proteins. To determine whether phosphorylation affected SPF45 binding to ΔFas RNA, we performed RIP analysis on COS-1 cells, which were cotransfected with ΔFas and wild-type or mutant Myc-SPF45. The ΔFas mRNA detected in anti-Myc immunoprecipitates was analysed by qRT-PCR and was shown to specifically interact with Myc-SPF45 (Figure 8B). Myc-SPF45-8A showed enhanced binding to ΔFas mRNA, whereas Myc-SPF45-8D showed reduced binding, with both reflecting the relative changes in exon 6 exclusion observed in the minigene splicing assays. These data suggest that SPF45 phosphorylation affects Fas mRNA binding.Figure 8.

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