Abstract
Purpose : PERP (p53 apoptosis effector related to PMP-22) is an apoptosis-specific p53 effector downregulated in uveal melanoma. This study aimed to investigate post translational modifications of PERP and evaluate their effect on PERP regulation in relation to induction of apoptosis.
Methods : Possible phosphorylation sites were predicted by in silico analysis and halo-tag system was used to generate Halo-PERP, Halo-PERP S46A and Halo-PERP S192A constructs. Halo-tagged proteins were pulled-down from transfected human melanoma mel202 cell lysate and analysed by immunoblotting alongside caspase 3 protein forms. Flow cytometry was used to evaluate apoptosis levels in transfected mel202 cells.
Results : Phosphorylation of serine residues of PERP was identified by specific immunoblotting. Two possible phospho-serine sites, S46 and S192, were identified by in silico analysis. Western blotting showed phosphorylated bands were maintained on PERP S192A-transfected mel202 cell lysates pull-downs and not on PERP S46A-transfectants. However, the apoptosis level determined by flow cytometry was lower in Halo-PERP S192A-expressing cells compared with Halo-PERP and Halo-PERP S46A-expressing cells, consistent also with the respective expression levels of caspase 3.
Conclusions : PERP is phosphorylated on S46 residue. However, this mutant does not appear to be involved in regulation of apoptosis. Instead, given its location in the extracellular loop, it is likely to be involved in the stability of the desmosome complex and therefore possibly linked to migratory properties of uveal melanoma cells. Intriguingly, PERP S192A mutant associates with decreased apoptosis suggesting its involvement in the p53 related apoptosis through a different mechanism.
Methods : Possible phosphorylation sites were predicted by in silico analysis and halo-tag system was used to generate Halo-PERP, Halo-PERP S46A and Halo-PERP S192A constructs. Halo-tagged proteins were pulled-down from transfected human melanoma mel202 cell lysate and analysed by immunoblotting alongside caspase 3 protein forms. Flow cytometry was used to evaluate apoptosis levels in transfected mel202 cells.
Results : Phosphorylation of serine residues of PERP was identified by specific immunoblotting. Two possible phospho-serine sites, S46 and S192, were identified by in silico analysis. Western blotting showed phosphorylated bands were maintained on PERP S192A-transfected mel202 cell lysates pull-downs and not on PERP S46A-transfectants. However, the apoptosis level determined by flow cytometry was lower in Halo-PERP S192A-expressing cells compared with Halo-PERP and Halo-PERP S46A-expressing cells, consistent also with the respective expression levels of caspase 3.
Conclusions : PERP is phosphorylated on S46 residue. However, this mutant does not appear to be involved in regulation of apoptosis. Instead, given its location in the extracellular loop, it is likely to be involved in the stability of the desmosome complex and therefore possibly linked to migratory properties of uveal melanoma cells. Intriguingly, PERP S192A mutant associates with decreased apoptosis suggesting its involvement in the p53 related apoptosis through a different mechanism.
| Original language | English |
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| Publication status | Published - 1 Jun 2022 |
| Event | 2022 ARVO Annual Meeting - event held in Denver and also virtually, Denver, United States Duration: 1 May 2022 → 4 May 2022 |
Conference
| Conference | 2022 ARVO Annual Meeting |
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| Country/Territory | United States |
| City | Denver |
| Period | 1/05/22 → 4/05/22 |