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Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.

Saddoughi SA, Gencer S, Peterson YK, Ward KE, Mukhopadhyay A, Oaks J, Bielawski J, Szulc ZM, Thomas RJ, Selvam SP, Senkal CE, Garrett-Mayer E, De Palma RM, Fedarovich D, Liu A, Habib AA, Stahelin RV, Perrotti D, Ogretmen B - EMBO Mol Med (2012)

Bottom Line: Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis.The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720.Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

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C18-ceramide selectively binds nuclear I2PP2A/SET through K209 and Y122 residuesA. I2PP2A/SET is docked to the hydrophobic domain of I2PP2A/SET, containing two anti-parallel beta-sheets and an alpha helix. Hydrophobic and hydrophilic residues are shown in red and blue, respectively.B. Molecular docking simulations of C18-ceramide to I2PP2A/SET identifies K209 residue interacting with the 1-OH group of ceramide. Close up examination of K209 residue of I2PP2A/SET shows a potential π-cationic interaction between K209 (cationic) and Y122 (π-arene) that may form a gate to the hydrophobic pocket of I2PP2A/SET.C. Sub-cellular localization of WT- and K209D-, Y122C- and ER-I2PP2A/SET-GFP were examined by confocal microscopy. Co-localization of GFP and calnexin (red) were determined for ER detection (yellow).D,E. Binding of WT-, K209D-, Y122C- and ER-I2PP2A/SET-GFP to endogenous C18-ceramide (D) versus C16-ceramide (E) was measured by LC/MS/MS. Samples were normalized to inorganic phosphate (Pi). Error bars show s.d., and **p < 0.01 were considered significant.
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fig01: C18-ceramide selectively binds nuclear I2PP2A/SET through K209 and Y122 residuesA. I2PP2A/SET is docked to the hydrophobic domain of I2PP2A/SET, containing two anti-parallel beta-sheets and an alpha helix. Hydrophobic and hydrophilic residues are shown in red and blue, respectively.B. Molecular docking simulations of C18-ceramide to I2PP2A/SET identifies K209 residue interacting with the 1-OH group of ceramide. Close up examination of K209 residue of I2PP2A/SET shows a potential π-cationic interaction between K209 (cationic) and Y122 (π-arene) that may form a gate to the hydrophobic pocket of I2PP2A/SET.C. Sub-cellular localization of WT- and K209D-, Y122C- and ER-I2PP2A/SET-GFP were examined by confocal microscopy. Co-localization of GFP and calnexin (red) were determined for ER detection (yellow).D,E. Binding of WT-, K209D-, Y122C- and ER-I2PP2A/SET-GFP to endogenous C18-ceramide (D) versus C16-ceramide (E) was measured by LC/MS/MS. Samples were normalized to inorganic phosphate (Pi). Error bars show s.d., and **p < 0.01 were considered significant.

Mentions: To uncover the structural details of I2PP2A/SET-ceramide binding, molecular modelling/simulations were performed using the crystal structure of I2PP2A/SET (Muto et al, 2007) and C18-ceramide as a probe (Fig 1A). Our previous study showed that a single mutation with K209D conversion significantly inhibited the binding of I2PP2A/SET to ceramide both in vitro and in A549 cells (Mukhopadhyay et al, 2009). Accordingly, one of the prominent docking sites of I2PP2A/SET for ceramide binding included the K209 residue (Fig 1A and B, and Supporting Information Fig S1), which interacts with the primary hydroxyl group of ceramide possibly via charge attraction (Fig 1B, and Supporting Information Fig S1). The model also suggested that the K209 directly interacts with the Y122 residue via a hydrophobic–ionic (cation/π-arene) interaction (Fig 1B), possibly playing a role as a gate for regulating the access of ceramide to the hydrophobic pocket.


Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.

Saddoughi SA, Gencer S, Peterson YK, Ward KE, Mukhopadhyay A, Oaks J, Bielawski J, Szulc ZM, Thomas RJ, Selvam SP, Senkal CE, Garrett-Mayer E, De Palma RM, Fedarovich D, Liu A, Habib AA, Stahelin RV, Perrotti D, Ogretmen B - EMBO Mol Med (2012)

C18-ceramide selectively binds nuclear I2PP2A/SET through K209 and Y122 residuesA. I2PP2A/SET is docked to the hydrophobic domain of I2PP2A/SET, containing two anti-parallel beta-sheets and an alpha helix. Hydrophobic and hydrophilic residues are shown in red and blue, respectively.B. Molecular docking simulations of C18-ceramide to I2PP2A/SET identifies K209 residue interacting with the 1-OH group of ceramide. Close up examination of K209 residue of I2PP2A/SET shows a potential π-cationic interaction between K209 (cationic) and Y122 (π-arene) that may form a gate to the hydrophobic pocket of I2PP2A/SET.C. Sub-cellular localization of WT- and K209D-, Y122C- and ER-I2PP2A/SET-GFP were examined by confocal microscopy. Co-localization of GFP and calnexin (red) were determined for ER detection (yellow).D,E. Binding of WT-, K209D-, Y122C- and ER-I2PP2A/SET-GFP to endogenous C18-ceramide (D) versus C16-ceramide (E) was measured by LC/MS/MS. Samples were normalized to inorganic phosphate (Pi). Error bars show s.d., and **p < 0.01 were considered significant.
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Related In: Results  -  Collection

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fig01: C18-ceramide selectively binds nuclear I2PP2A/SET through K209 and Y122 residuesA. I2PP2A/SET is docked to the hydrophobic domain of I2PP2A/SET, containing two anti-parallel beta-sheets and an alpha helix. Hydrophobic and hydrophilic residues are shown in red and blue, respectively.B. Molecular docking simulations of C18-ceramide to I2PP2A/SET identifies K209 residue interacting with the 1-OH group of ceramide. Close up examination of K209 residue of I2PP2A/SET shows a potential π-cationic interaction between K209 (cationic) and Y122 (π-arene) that may form a gate to the hydrophobic pocket of I2PP2A/SET.C. Sub-cellular localization of WT- and K209D-, Y122C- and ER-I2PP2A/SET-GFP were examined by confocal microscopy. Co-localization of GFP and calnexin (red) were determined for ER detection (yellow).D,E. Binding of WT-, K209D-, Y122C- and ER-I2PP2A/SET-GFP to endogenous C18-ceramide (D) versus C16-ceramide (E) was measured by LC/MS/MS. Samples were normalized to inorganic phosphate (Pi). Error bars show s.d., and **p < 0.01 were considered significant.
Mentions: To uncover the structural details of I2PP2A/SET-ceramide binding, molecular modelling/simulations were performed using the crystal structure of I2PP2A/SET (Muto et al, 2007) and C18-ceramide as a probe (Fig 1A). Our previous study showed that a single mutation with K209D conversion significantly inhibited the binding of I2PP2A/SET to ceramide both in vitro and in A549 cells (Mukhopadhyay et al, 2009). Accordingly, one of the prominent docking sites of I2PP2A/SET for ceramide binding included the K209 residue (Fig 1A and B, and Supporting Information Fig S1), which interacts with the primary hydroxyl group of ceramide possibly via charge attraction (Fig 1B, and Supporting Information Fig S1). The model also suggested that the K209 directly interacts with the Y122 residue via a hydrophobic–ionic (cation/π-arene) interaction (Fig 1B), possibly playing a role as a gate for regulating the access of ceramide to the hydrophobic pocket.

Bottom Line: Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis.The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720.Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

Show MeSH
Related in: MedlinePlus