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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-I2PP2A/SET binding activates PP2AA. Knockdown of I2PP2A/SET using shRNAs and reconstitution of WT-I2PP2A compared to controls in A549 cells were confirmed by Western blotting. Actin was used a loading control.B. PP2A activity assay was performed using extracts obtained from A549, A549/shI2PP2A and A549/shI2PP2A/WT-I2PP2A/SET grown in the absence/presence of 10 µM FTY720.C. Effects of FTY720 (20 µM) on cell death was measured by detection of LDH secretion in the absence/presence of OA (10 nM).D,E. Roles of si-RNA-mediated PP2A knockdown (confirmed by Western blotting, shown in left panel) in the modulation of PP2A activity (D) or FTY720-mediated cell death (E) were measured in A549 cells compared to Scr-siRNA controls (right panel).F. Effects of PP2A-HA expression on cell death in the absence/presence of FTY720 was measured compared to controls. Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.
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fig06: FTY720-I2PP2A/SET binding activates PP2AA. Knockdown of I2PP2A/SET using shRNAs and reconstitution of WT-I2PP2A compared to controls in A549 cells were confirmed by Western blotting. Actin was used a loading control.B. PP2A activity assay was performed using extracts obtained from A549, A549/shI2PP2A and A549/shI2PP2A/WT-I2PP2A/SET grown in the absence/presence of 10 µM FTY720.C. Effects of FTY720 (20 µM) on cell death was measured by detection of LDH secretion in the absence/presence of OA (10 nM).D,E. Roles of si-RNA-mediated PP2A knockdown (confirmed by Western blotting, shown in left panel) in the modulation of PP2A activity (D) or FTY720-mediated cell death (E) were measured in A549 cells compared to Scr-siRNA controls (right panel).F. Effects of PP2A-HA expression on cell death in the absence/presence of FTY720 was measured compared to controls. Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.

Mentions: To examine whether binding/targeting of I2PP2A/SET by FTY720 inhibits lung tumour growth via PP2A activation, we measured PP2A activity in response to shRNA-mediated I2PP2A knockdown (∼90%) in A549 cells (Fig 6A). Then, effects of WT-I2PP2A/SET reconstitution (∼60%) in A549/sh-I2PP2A/SET cells on PP2A activity in response to FTY720 was also measured compared to controls (Fig 6A). Interestingly, whereas FTY720 enhanced PP2A activity compared to vehicle-treated controls, knockdown of I2PP2A/SET also significantly enhanced PP2A activity, but prevented its further activation by FTY720 (Fig 6B). In contrast, when WT-I2PP2A/SET was restored in A549/sh-I2PP2A/SET cells, the PP2A activity was increased in response to FTY720 (Fig 6B). Thus, these data suggest that FTY720 activates PP2A via targeting I2PP2A/SET.


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-I2PP2A/SET binding activates PP2AA. Knockdown of I2PP2A/SET using shRNAs and reconstitution of WT-I2PP2A compared to controls in A549 cells were confirmed by Western blotting. Actin was used a loading control.B. PP2A activity assay was performed using extracts obtained from A549, A549/shI2PP2A and A549/shI2PP2A/WT-I2PP2A/SET grown in the absence/presence of 10 µM FTY720.C. Effects of FTY720 (20 µM) on cell death was measured by detection of LDH secretion in the absence/presence of OA (10 nM).D,E. Roles of si-RNA-mediated PP2A knockdown (confirmed by Western blotting, shown in left panel) in the modulation of PP2A activity (D) or FTY720-mediated cell death (E) were measured in A549 cells compared to Scr-siRNA controls (right panel).F. Effects of PP2A-HA expression on cell death in the absence/presence of FTY720 was measured compared to controls. Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.
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fig06: FTY720-I2PP2A/SET binding activates PP2AA. Knockdown of I2PP2A/SET using shRNAs and reconstitution of WT-I2PP2A compared to controls in A549 cells were confirmed by Western blotting. Actin was used a loading control.B. PP2A activity assay was performed using extracts obtained from A549, A549/shI2PP2A and A549/shI2PP2A/WT-I2PP2A/SET grown in the absence/presence of 10 µM FTY720.C. Effects of FTY720 (20 µM) on cell death was measured by detection of LDH secretion in the absence/presence of OA (10 nM).D,E. Roles of si-RNA-mediated PP2A knockdown (confirmed by Western blotting, shown in left panel) in the modulation of PP2A activity (D) or FTY720-mediated cell death (E) were measured in A549 cells compared to Scr-siRNA controls (right panel).F. Effects of PP2A-HA expression on cell death in the absence/presence of FTY720 was measured compared to controls. Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.
Mentions: To examine whether binding/targeting of I2PP2A/SET by FTY720 inhibits lung tumour growth via PP2A activation, we measured PP2A activity in response to shRNA-mediated I2PP2A knockdown (∼90%) in A549 cells (Fig 6A). Then, effects of WT-I2PP2A/SET reconstitution (∼60%) in A549/sh-I2PP2A/SET cells on PP2A activity in response to FTY720 was also measured compared to controls (Fig 6A). Interestingly, whereas FTY720 enhanced PP2A activity compared to vehicle-treated controls, knockdown of I2PP2A/SET also significantly enhanced PP2A activity, but prevented its further activation by FTY720 (Fig 6B). In contrast, when WT-I2PP2A/SET was restored in A549/sh-I2PP2A/SET cells, the PP2A activity was increased in response to FTY720 (Fig 6B). Thus, these data suggest that FTY720 activates PP2A via targeting I2PP2A/SET.

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