Protein Phosphatase 4 Regulatory Subunit 3A regulates leukemic cell survival independent of PP4 catalytic subunit


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Mirna Mourtada-Maarabouni1,Nadieh Kavousi2
1Keele University,2University of Keele

Abstract

Background

The serine/ threonine protein phosphatase 4 holoenzyme consists of PP4 catalytic subunit (PP4c) which interacts with four different regulatory subunits (PP4R1, PP4R2, PP4R3, PP4R4). We have previously showed that PP4c acts as a tumour suppressor and control cell fate of both leukemic T-cells and untransformed human peripheral blood T-cells. The present work examined the effects of modulation  PP4 regulatory subunit 3A (PP4R3A/ SMEK1) expression on apoptosis and cell proliferation in human leukemic T-cell lines. We have also investigated the involvement of PP4R3A in mediating the effects of PP4c.

Method

The human leukemic T-cell lines Jurkat and CEM-C7 were transfected with pcDNA3.1-PP4R3A or PP4R3A specific siRNAs and controls received empty vector or scrambled siRNA, as appropriate. Endogenous expression level of PP4c and PP4R3A was confirmed by western blotting. Culture growth, cell viability, apoptosis and cell cycle were assessed using flow cytometry. To investigate the role of PP4R3A in mediating the effects of PP4c, cells were transfected with three different PP4R3A siRNAs or PP4C siRNAs, control cells received scrambled siRNA. 48 h post-transfection, control cells (transfected with negative control siRNA) and cells transfected with PP4R3A siRNAs or PP4C siRNAs were transiently transfected with pcDNA3.1-PP4c or pcDNA3.1-PP4R3A  expression construct (as appropriate) or pcDNA3.1. Cell viability and apoptosis level were assessed post transfection.

Results

PP4R3A overexpression decreased cell growth, increased spontaneous apoptosis rate, reduced colony forming ability and affected cell cycle progression. Down-regulation of PP4C  did not alter the pro-apoptotic effects of PP4R3A. On the other hand, cells transfected with PP4R3A siRNAs showed an increase in the survival rate and reduction in the level of spontaneous apoptosis. Down-regulation of PP4R3A did not reverse the pro-apoptotic effects of PP4C.

Conclusion

PP4R3A or SMEK1 regulates cell survival and proliferation in leukemic T-cells. PP4R3A appears to function independently from PP4c and has no role in mediating PP4C effects. The results suggest that PP4R3A function as a cell fate-regulating protein and its dysfunction may be important in the development and progression of leukemia.