Limits...
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.

Show MeSH

Related in: MedlinePlus

SPF45 overexpression enhances fibronectin expression in a phosphorylation site-specific manner. (A) Fibronectin mRNA expression was measured in stable SKOV-3 cells by qRT-PCR. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group. (B) Protein lysates from cells in (A) were immunoblotted with anti-fibronectin (FN) and anti-actin antibodies. (C) Fibronectin protein levels in OV2008 stable cell lines were determined by immunoblotting as in (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643583&req=5

gkt170-F11: SPF45 overexpression enhances fibronectin expression in a phosphorylation site-specific manner. (A) Fibronectin mRNA expression was measured in stable SKOV-3 cells by qRT-PCR. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group. (B) Protein lysates from cells in (A) were immunoblotted with anti-fibronectin (FN) and anti-actin antibodies. (C) Fibronectin protein levels in OV2008 stable cell lines were determined by immunoblotting as in (B).

Mentions: Fibronectin enhances cell migration (43–46), and we previously showed that SPF45 overexpression enhanced fibronectin expression in SKOV-3 cells in a manner independent of SPF45 phosphorylation by MAP kinases (21). Stable expression of SPF45 or SPF45-8D in SKOV-3 cells increased fibronectin mRNA and protein expression, whereas SPF45-8A expression completely inhibited fibronectin expression (Figure 11A and B), indicating that SPF45 regulates fibronectin expression in a Clk1 phosphorylation-dependent manner. In OV2008 cells, SPF45 and SPF45-8D overexpression also increased fibronectin protein levels compared with vector cell and cells expressing SPF45-8A (Figure 11C).Figure 11.


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 enhances fibronectin expression in a phosphorylation site-specific manner. (A) Fibronectin mRNA expression was measured in stable SKOV-3 cells by qRT-PCR. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group. (B) Protein lysates from cells in (A) were immunoblotted with anti-fibronectin (FN) and anti-actin antibodies. (C) Fibronectin protein levels in OV2008 stable cell lines were determined by immunoblotting as in (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643583&req=5

gkt170-F11: SPF45 overexpression enhances fibronectin expression in a phosphorylation site-specific manner. (A) Fibronectin mRNA expression was measured in stable SKOV-3 cells by qRT-PCR. Results from six experiments are shown as mean ± SE. *P < 0.05 versus vec group, #P < 0.05 versus wt group. (B) Protein lysates from cells in (A) were immunoblotted with anti-fibronectin (FN) and anti-actin antibodies. (C) Fibronectin protein levels in OV2008 stable cell lines were determined by immunoblotting as in (B).
Mentions: Fibronectin enhances cell migration (43–46), and we previously showed that SPF45 overexpression enhanced fibronectin expression in SKOV-3 cells in a manner independent of SPF45 phosphorylation by MAP kinases (21). Stable expression of SPF45 or SPF45-8D in SKOV-3 cells increased fibronectin mRNA and protein expression, whereas SPF45-8A expression completely inhibited fibronectin expression (Figure 11A and B), indicating that SPF45 regulates fibronectin expression in a Clk1 phosphorylation-dependent manner. In OV2008 cells, SPF45 and SPF45-8D overexpression also increased fibronectin protein levels compared with vector cell and cells expressing SPF45-8A (Figure 11C).Figure 11.

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