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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.

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

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FTY720 binds I2PP2A/SETA. Binding of endogenous sphingosine was detected by LC/MS/MS in A549 lung cancer cells after overexpression and pull-down of vector, WT-I2PP2A or K209D-I2PP2A-GFP, using anti-GFP columns.B. Molecular docking predicts various residues of I2PP2A/SET that may be involved in FTY720 binding, including K209 and Y122. The 1-OH of FTY720 is expected to interact with the K209, which might form a cationic/π-arene interaction with the Y122 to form a possible gate for FTY720/ceramide binding.C. Binding of I2PP2A/SET to B-FTY720 (1, 5 or 10 µM) was detected using avidin pull-down (elution) followed by Western blotting with the anti-I2PP2A/SET antibody. Structure of B-FTY720 is shown. Full blots can be seen in Supporting Information Fig S15.D. Binding of B-FTY720 (10 µM) to WT-I2PP2A/SET compared to K209D- or Y122C-I2PP2A-GFP was measured as described in Materials and Methods section.E,F. Binding of purified human I2PP2A/SET to C18-ceramide and FTY720 (E) or P-FTY720 (F) were measured using SPR. Saturation values determined for 1 µM I2PP2A/SET injected over the respective active surface. The average response for 1 µM I2PP2A/SET binding to C18-ceramide, FTY720 or P-FTY720 over the control POPC:POPE surface, performed in triplicate, were shown. Error bars represent s.d. (p < 0.05, p < 0.01, p < 0.001 were considered significant).
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fig03: FTY720 binds I2PP2A/SETA. Binding of endogenous sphingosine was detected by LC/MS/MS in A549 lung cancer cells after overexpression and pull-down of vector, WT-I2PP2A or K209D-I2PP2A-GFP, using anti-GFP columns.B. Molecular docking predicts various residues of I2PP2A/SET that may be involved in FTY720 binding, including K209 and Y122. The 1-OH of FTY720 is expected to interact with the K209, which might form a cationic/π-arene interaction with the Y122 to form a possible gate for FTY720/ceramide binding.C. Binding of I2PP2A/SET to B-FTY720 (1, 5 or 10 µM) was detected using avidin pull-down (elution) followed by Western blotting with the anti-I2PP2A/SET antibody. Structure of B-FTY720 is shown. Full blots can be seen in Supporting Information Fig S15.D. Binding of B-FTY720 (10 µM) to WT-I2PP2A/SET compared to K209D- or Y122C-I2PP2A-GFP was measured as described in Materials and Methods section.E,F. Binding of purified human I2PP2A/SET to C18-ceramide and FTY720 (E) or P-FTY720 (F) were measured using SPR. Saturation values determined for 1 µM I2PP2A/SET injected over the respective active surface. The average response for 1 µM I2PP2A/SET binding to C18-ceramide, FTY720 or P-FTY720 over the control POPC:POPE surface, performed in triplicate, were shown. Error bars represent s.d. (p < 0.05, p < 0.01, p < 0.001 were considered significant).

Mentions: Targeting I2PP2A/SET exogenously with a sphingolipid analogue drug that mimics ceramide and/or sphingosine could potentially bind I2PP2A/SET and reactivate PP2A tumour suppressor signalling (Mukhopadhyay et al, 2009). Since exogenous (Mukhopadhyay et al, 2009) and endogenous sphingosine (Fig 3A) bind wt-I2PP2A/SET, but not K209D-I2PP2A/SET, in A549 cells (Fig 3A), we examined whether sphingosine analogue FTY720, which was shown to inhibit tumour growth (Liu et al, 2010; Neviani et al, 2007; Pchejetski et al, 2010; Wallington-Beddoe et al, 2011), binds/targets I2PP2A/SET. We performed molecular docking studies based on I2PP2A/SET–ceramide modelling (Fig 1A and B), which predicted that one of the primary hydroxyl groups of FTY720 might bind to the K209 residue of I2PP2A/SET (Fig 3B and Supporting Information Fig S7). Biotin-labelled FTY720 (B-FTY720) was incubated with purified I2PP2A/SET, drug-bound proteins were then separated through an avidin column, and B-FTY720-bound I2PP2A/SET was detected by Western blotting using anti-I2PP2A/SET antibody. Remarkably, FTY720 (5–10 µM) also bound directly to purified I2PP2A/SET in vitro or endogenously expressed I2PP2A/SET in A549 cells (Fig 3C).


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)

FTY720 binds I2PP2A/SETA. Binding of endogenous sphingosine was detected by LC/MS/MS in A549 lung cancer cells after overexpression and pull-down of vector, WT-I2PP2A or K209D-I2PP2A-GFP, using anti-GFP columns.B. Molecular docking predicts various residues of I2PP2A/SET that may be involved in FTY720 binding, including K209 and Y122. The 1-OH of FTY720 is expected to interact with the K209, which might form a cationic/π-arene interaction with the Y122 to form a possible gate for FTY720/ceramide binding.C. Binding of I2PP2A/SET to B-FTY720 (1, 5 or 10 µM) was detected using avidin pull-down (elution) followed by Western blotting with the anti-I2PP2A/SET antibody. Structure of B-FTY720 is shown. Full blots can be seen in Supporting Information Fig S15.D. Binding of B-FTY720 (10 µM) to WT-I2PP2A/SET compared to K209D- or Y122C-I2PP2A-GFP was measured as described in Materials and Methods section.E,F. Binding of purified human I2PP2A/SET to C18-ceramide and FTY720 (E) or P-FTY720 (F) were measured using SPR. Saturation values determined for 1 µM I2PP2A/SET injected over the respective active surface. The average response for 1 µM I2PP2A/SET binding to C18-ceramide, FTY720 or P-FTY720 over the control POPC:POPE surface, performed in triplicate, were shown. Error bars represent s.d. (p < 0.05, p < 0.01, p < 0.001 were considered significant).
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fig03: FTY720 binds I2PP2A/SETA. Binding of endogenous sphingosine was detected by LC/MS/MS in A549 lung cancer cells after overexpression and pull-down of vector, WT-I2PP2A or K209D-I2PP2A-GFP, using anti-GFP columns.B. Molecular docking predicts various residues of I2PP2A/SET that may be involved in FTY720 binding, including K209 and Y122. The 1-OH of FTY720 is expected to interact with the K209, which might form a cationic/π-arene interaction with the Y122 to form a possible gate for FTY720/ceramide binding.C. Binding of I2PP2A/SET to B-FTY720 (1, 5 or 10 µM) was detected using avidin pull-down (elution) followed by Western blotting with the anti-I2PP2A/SET antibody. Structure of B-FTY720 is shown. Full blots can be seen in Supporting Information Fig S15.D. Binding of B-FTY720 (10 µM) to WT-I2PP2A/SET compared to K209D- or Y122C-I2PP2A-GFP was measured as described in Materials and Methods section.E,F. Binding of purified human I2PP2A/SET to C18-ceramide and FTY720 (E) or P-FTY720 (F) were measured using SPR. Saturation values determined for 1 µM I2PP2A/SET injected over the respective active surface. The average response for 1 µM I2PP2A/SET binding to C18-ceramide, FTY720 or P-FTY720 over the control POPC:POPE surface, performed in triplicate, were shown. Error bars represent s.d. (p < 0.05, p < 0.01, p < 0.001 were considered significant).
Mentions: Targeting I2PP2A/SET exogenously with a sphingolipid analogue drug that mimics ceramide and/or sphingosine could potentially bind I2PP2A/SET and reactivate PP2A tumour suppressor signalling (Mukhopadhyay et al, 2009). Since exogenous (Mukhopadhyay et al, 2009) and endogenous sphingosine (Fig 3A) bind wt-I2PP2A/SET, but not K209D-I2PP2A/SET, in A549 cells (Fig 3A), we examined whether sphingosine analogue FTY720, which was shown to inhibit tumour growth (Liu et al, 2010; Neviani et al, 2007; Pchejetski et al, 2010; Wallington-Beddoe et al, 2011), binds/targets I2PP2A/SET. We performed molecular docking studies based on I2PP2A/SET–ceramide modelling (Fig 1A and B), which predicted that one of the primary hydroxyl groups of FTY720 might bind to the K209 residue of I2PP2A/SET (Fig 3B and Supporting Information Fig S7). Biotin-labelled FTY720 (B-FTY720) was incubated with purified I2PP2A/SET, drug-bound proteins were then separated through an avidin column, and B-FTY720-bound I2PP2A/SET was detected by Western blotting using anti-I2PP2A/SET antibody. Remarkably, FTY720 (5–10 µM) also bound directly to purified I2PP2A/SET in vitro or endogenously expressed I2PP2A/SET in A549 cells (Fig 3C).

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