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C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1.

Sumanasekera C, Kelemen O, Beullens M, Aubol BE, Adams JA, Sunkara M, Morris A, Bollen M, Andreadis A, Stamm S - Nucleic Acids Res. (2011)

Bottom Line: To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection.We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF).Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.

ABSTRACT
Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

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C6 pyridinium ceramide inhibits protein phosphatase-1. (A) An equimolar mixture of PP1 isoforms (alpha, beta and gamma) was incubated with the concentrations of C6 pyrimidinim ceramide indicated and the activity on p-nitrophenylphosphate was determined. 1-(2-Oxopropyl) pyridinium was used as a negative control. (B) The effect on the PP1/PP2A inhibitor calyculin A is shown for comparison. (C) PyrCer effect on PP1 dephosphorylating phosphorylase phosphatase. (D) PyrCer effect on the phophorylation of splicing factors. HeLa nuclear extract was incubated with [γ-32P] ATP with or without 10 µM PyrCer and the proteins indicated were immunoprecipated. The top lanes (32P) show the signal from overnight autoradiography. The signal form western blot using the antisera employed in immunorecipitations are shown below. Tra2-beta1 and PSF migrate as doublet in western blots, which is due to different phosphorylation states (24). The numbers indicate the molecular weights. (E) Effect of 10 µM PyrCer on PP1-mediated dephosphorylation of purified Tra2-beta1 in vitro.
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gkr1289-F3: C6 pyridinium ceramide inhibits protein phosphatase-1. (A) An equimolar mixture of PP1 isoforms (alpha, beta and gamma) was incubated with the concentrations of C6 pyrimidinim ceramide indicated and the activity on p-nitrophenylphosphate was determined. 1-(2-Oxopropyl) pyridinium was used as a negative control. (B) The effect on the PP1/PP2A inhibitor calyculin A is shown for comparison. (C) PyrCer effect on PP1 dephosphorylating phosphorylase phosphatase. (D) PyrCer effect on the phophorylation of splicing factors. HeLa nuclear extract was incubated with [γ-32P] ATP with or without 10 µM PyrCer and the proteins indicated were immunoprecipated. The top lanes (32P) show the signal from overnight autoradiography. The signal form western blot using the antisera employed in immunorecipitations are shown below. Tra2-beta1 and PSF migrate as doublet in western blots, which is due to different phosphorylation states (24). The numbers indicate the molecular weights. (E) Effect of 10 µM PyrCer on PP1-mediated dephosphorylation of purified Tra2-beta1 in vitro.

Mentions: It has been previously reported that ceramides promote PP1 activity (13) and that exogenous d-(e)-C6-ceramide causes SR-protein dephosphorylation (24). We therefore tested whether the PyrCer has an influence on PP1 activity using the classical p-nitrophenyl phosphate phosphatase assay. An equimolar mixture of all three PP1 isoforms purified from rabbit was used as the PP1 source. As shown in Figure 3A, PyrCer blocks PP1 activity at 100 µM concentration, when 0.1 U PP1 are present. In contrast, oxo-pyridinium that represents the water-soluble moiety of PyrCer had no significant effect. We used calyculin A as a positive control for a PP1 inhibitor and found that it inhibits PP1 activity at a 10 nM concentration (Figure 3A and B). We next tested the influence of PyrCer on the natural PP1 substrate phosphorylase phosphatase and again found an inhibition starting from 25 µM (Figure 3C).Figure 3.


C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1.

Sumanasekera C, Kelemen O, Beullens M, Aubol BE, Adams JA, Sunkara M, Morris A, Bollen M, Andreadis A, Stamm S - Nucleic Acids Res. (2011)

C6 pyridinium ceramide inhibits protein phosphatase-1. (A) An equimolar mixture of PP1 isoforms (alpha, beta and gamma) was incubated with the concentrations of C6 pyrimidinim ceramide indicated and the activity on p-nitrophenylphosphate was determined. 1-(2-Oxopropyl) pyridinium was used as a negative control. (B) The effect on the PP1/PP2A inhibitor calyculin A is shown for comparison. (C) PyrCer effect on PP1 dephosphorylating phosphorylase phosphatase. (D) PyrCer effect on the phophorylation of splicing factors. HeLa nuclear extract was incubated with [γ-32P] ATP with or without 10 µM PyrCer and the proteins indicated were immunoprecipated. The top lanes (32P) show the signal from overnight autoradiography. The signal form western blot using the antisera employed in immunorecipitations are shown below. Tra2-beta1 and PSF migrate as doublet in western blots, which is due to different phosphorylation states (24). The numbers indicate the molecular weights. (E) Effect of 10 µM PyrCer on PP1-mediated dephosphorylation of purified Tra2-beta1 in vitro.
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Related In: Results  -  Collection

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gkr1289-F3: C6 pyridinium ceramide inhibits protein phosphatase-1. (A) An equimolar mixture of PP1 isoforms (alpha, beta and gamma) was incubated with the concentrations of C6 pyrimidinim ceramide indicated and the activity on p-nitrophenylphosphate was determined. 1-(2-Oxopropyl) pyridinium was used as a negative control. (B) The effect on the PP1/PP2A inhibitor calyculin A is shown for comparison. (C) PyrCer effect on PP1 dephosphorylating phosphorylase phosphatase. (D) PyrCer effect on the phophorylation of splicing factors. HeLa nuclear extract was incubated with [γ-32P] ATP with or without 10 µM PyrCer and the proteins indicated were immunoprecipated. The top lanes (32P) show the signal from overnight autoradiography. The signal form western blot using the antisera employed in immunorecipitations are shown below. Tra2-beta1 and PSF migrate as doublet in western blots, which is due to different phosphorylation states (24). The numbers indicate the molecular weights. (E) Effect of 10 µM PyrCer on PP1-mediated dephosphorylation of purified Tra2-beta1 in vitro.
Mentions: It has been previously reported that ceramides promote PP1 activity (13) and that exogenous d-(e)-C6-ceramide causes SR-protein dephosphorylation (24). We therefore tested whether the PyrCer has an influence on PP1 activity using the classical p-nitrophenyl phosphate phosphatase assay. An equimolar mixture of all three PP1 isoforms purified from rabbit was used as the PP1 source. As shown in Figure 3A, PyrCer blocks PP1 activity at 100 µM concentration, when 0.1 U PP1 are present. In contrast, oxo-pyridinium that represents the water-soluble moiety of PyrCer had no significant effect. We used calyculin A as a positive control for a PP1 inhibitor and found that it inhibits PP1 activity at a 10 nM concentration (Figure 3A and B). We next tested the influence of PyrCer on the natural PP1 substrate phosphorylase phosphatase and again found an inhibition starting from 25 µM (Figure 3C).Figure 3.

Bottom Line: To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection.We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF).Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.

ABSTRACT
Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

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