TI - Discussion . AB - Here we report that PP4c exerts profound and specific effects on the growth and survival of both normal and leukemic human T-cells . The data presented reveal that over-expression of PP4c in the T-leukemic cell lines CEM-C7 and Jurkat inhibited their proliferation in the absence of extracellular apoptotic stimuli by inducing apoptosis and cell cycle arrest in G1 . Our work also showed that down-regulation of endogenous PP4c by 60-70% increased the rate of cell proliferation and conferred resistance to a number of apoptotic stimuli . While down-regulation of PP4c conferred substantial resistance to UV , cisplatin and butyrate , it protected against Fas -induced apoptosis only partially . This partial inhibition of Fas -induced apoptosis caused by down-regulation of PP4 may be explained in part by recognizing that , in addition to caspase -dependent pathways , Fas ligation activates other major signaling cascades that belong to the family of mitogen-activated protein kinase ( MAPK ) pathways [52,53] . Down-regulation of PP4c protected against Dexamethasone -induced apoptosis but was less effective in protecting against the loss of colony-forming ability . This may indicate that PP4c down-regulation protects only against dexamethasone-induced apoptosis and is less effective against the anti-proliferative effects of dexamethasone . These results suggest that PP4 may be able to modulate apoptosis in leukemic T-cells via a common factor which affects the apoptosis signaling pathways mediated by these different apoptotic stimuli . The modulation of PP4c level did not affect cell death induced by okadaic/ acid , presumably because okadaic/ acid also inhibits other phosphatases , particularly PP2 , with a comparable IC50[55] . The critical importance of PP4c levels for the growth and survival of T-cells was further demonstrated using primary cultures of PHA -stimulated human peripheral blood lymphocytes . As for the leukemic T-cell populations , our data show that over-expression of PP4c caused inhibition of normal lymphocyte proliferation and an increase in apoptosis , whereas down-regulation of PP4c was accompanied by a corresponding increase in proliferation and inhibition of apoptosis . Our results do not conflict with previous work which showed that PP4c activity is required for microtubule nucleation during centrosome maturation in Caenorhabditis elegans[29] , and for the development of thymocytes in mice and to have an anti-apoptotic role in thymocytes [42] , since these observations can quickly be reconciled by recognition of the wide range of cellular processes which involve PP4c . It is therefore likely that a minimum level of this enzyme is required for survival and proliferation . Since our analysis was inevitably focused on viable cells capable of replication and colony formation , we would not have detected any cells where the PP4 activity was below the minimum required for centrosome maturation . Recent studies have shown that PP4 regulates an increasing number of cellular functions in different cellular locations and have resulted in the identification of several PP4 regulatory subunits and binding proteins [22 , 24 , 29 , 31] . Like other serine/threonine phosphatases , PP4 is probably targeted to its specific sites of action by these regulatory subunits and it is likely that the interchange between the different regulatory subunits and binding proteins plays a critical role in regulating the activity of PP4 complexes . It is also important to note that PP4c is a positive regulator of HPK1 [38] , which in turn is known to promote apoptosis of murine T lymphocytes [57,58] . The possibility that over-expression of PP4c promotes apoptosis via the activation of HPK1 therefore requires further investigation . It is also believed that loss of DNA repair systems that prevent the fixation of premutagenic lesions in the genome is mandatory for carcinogenesis , since the increased mutation rates of unstable cells can give them a growth advantage over normal cells and allow them to accumulate aberrations that subsequently lead to cancer [59] . In the present study , we monitored the rate of mutation at the HPRT marker locus in order to study the effects of PP4c modulation on the mutation rate of leukemic T-cells . In agreement with our previous studies using HEK 293T cells [41] , our results showed that over-expression of PP4c significantly reduces mutation at the HPRT locus . These effects were dependent on PP4c catalytic activity since the PP4c phosphatase-dead mutant ( PP4-RL ) did not affect the mutation rate of these cells ( results not shown ) . On the other hand , down regulation of endogenous PP4c consistently increased the HPRT gene mutation frequency . These observations have important implications for oncogenesis and further support an important rate-limiting role for PP4c in the induction of apoptosis after DNA damage . PP4 shares 65% amino acid identity with PP2 , the most abundant serine/threonine phosphatase in mammals [23,24] , which plays a crucial role in many fundamental processes including differentiation , embryonic development and growth control [60,61] . The great versatility of PP2 is a result of the existence of a large number of subunits : two isoforms of the catalytic C subunit ( Calpha and Cbeta ) , two isoforms of the regulatory/scaffolding A subunit ( Aalpha and Abeta ) and numerous regulatory B subunits that fall into four families designated B , B' , B'' and B '''[62-66] . In addition , PP2 , PP4 and PP6 share the alpha 4 ( alpha4 ) protein , the mammalian ortholog of yeast Tap42 , which binds to the catalytic subunits and displaces other regulatory subunits [28,67] . Several lines of evidence implicate PP2 in the regulation of apoptosis [28,43,44,61,68] . However , as for PP4 , the reported effects of PP2 on apoptosis and cell growth appear variable , since its function has been variously described as pro-apoptotic [69,70] and as anti-apoptotic [71] . Functional screens of human phosphatases have identified the alpha isoform of PP2 catalytic subunit as associated with survival and the beta isoform as associated with cell death [51] . Our data indicate that PP2cbeta over-expression causes an inhibitory effect on cell proliferation and enhances apoptosis , producing effects similar to those observed with PP4c in these cells . In addition , as with the knockdown of PP4c , down-regulation of PP2cbeta increased cell proliferation . However , while PP4c knock-down conferred resistance to fetal calf serum withdrawal , reducing PP2cbeta levels had little effect in this situation , indicating that each of these phosphatases acts independently on different apoptosis pathways . alpha4 protein , a PP2 - and PP4-regulatory subunit , has been implicated in the regulation of B - and T-cell differentiation , embryonic development and cell death [71-73] . Knockout of alpha4 decreases cell proliferation and promotes apoptosis in thymocytes as well as in other cell types [74,75] . alpha4 has been shown to interact directly with PP6 , PP4c and PP2 catalytic subunits and exerts opposing kinetic effects on these target phosphatases [28] . It is likely that the pro-apoptotic effects observed as a result of alpha4 knockdown are produced by the increased activity of PP4c and/or the inhibition or activation of PP2 catalytic subunits . Further work is required to investigate the interplay between PP4c , PP2 catalytic subunit isoforms and alpha4 and to determine their roles in commitment to apoptosis . A multiplexed phosphorylaTION screen revealed that modulation of the endogenous level of PP4c in HEK 293T cells resulted in an increase or reduction in the PHOSphorylation state of many proteins involved in cellular stress , cellular proliferation and apoptosis [41] . The PHOSphorylation of PEA-15 was found to increase dramatically ( by 223% ) when PP4c expression was reduced [41] . PEA-15 has been reported to modulate signalling pathways that control apoptosis and cell proliferation [45,46] and to play a rate-limiting role in the induction of B-CLL cell apoptosis induced by TRAIL [47] . PEA-15 can act to modulate apoptosis and as a critical regulator of the cell cycle . Its function is tightly regulated by PHOSphorylation and is involved in the signalling pathways mediated by ERK1/2 , Akt and RSK2 [45 , 46 , 56 , 76 , 77] . In the present study , we have investigated the involvement of PEA-15 in mediating the effects of PP4c on T-leukemic cells . Down-regulation of PEA-15 reduced viable cell number , in accordance with its reported effects as an anti-apoptotic protein . PP4c over-expression in the cells which have down-regulated PEA-15 did not cause additional cell death , in contrast to the effects of PP2cbeta over-expression . These observations suggest that the induction of apoptosis by over-expression of PP4c is mediated , atleast in part , by the dePHOSphorylation of PEA-15 . This highlights clear differences between the pathways which mediate the effects of PP4c and those that mediate the effects of PP2 , which appear to be largely independent of PEA-15 . Down-regulation of PP4c was able to partially reverse the inhibitory effects of PEA-15 knockdown , suggesting that PP4c activity is normally an important element in regulating PEA-15 activity . The interaction between PEA-15 and PP4c may therefore be critical in leukemogenesis and leukemia progression . The PEA-15 gene is amplified in breast cancer as well as in other cancers [78] , where PEA-15 over-expression confers resistance to abroad range of anti-cancer drugs [79] . Since PEA-15 also confers TRAIL-resistance on leukemic cells [47] , and the reistance conferred depends on the PHOSphorylation status of PEA-15 [54] , PP4 activity is likely to play an important role in regulating leukemic cell drug sensitivity . Akt , which PHOSphorylates and stabilises the anti-apoptotic action of PEA-15 , is also upregulated in a number of human cancers [80] , suggesting that they might function cooperatively in tumorigenesis . Developing strategies that enhance the activity of PP4c to oppose the effects Akt on PEA-15 could therefore prove to be effective in treating cancer .