Inhibition of Protein Phosphatase 2A (PP2A) Prevents Mcl-1 Protein Dephosphorylation at the Thr-163/Ser-159 Phosphodegron, Dramatically Reducing Expression in Mcl-1-amplified Lymphoma Cells*

Background: Although Mcl-1 is normally subject to rapid turnover via a phosphodegron at Thr-163/Ser-159 and other pathways, sustained expression promotes viability/chemoresistance in cancer cells. Results: Inhibition or knockdown of PP2A increases phosphorylation at Thr-163 and Ser-159 while decreasing Mcl-1 expression. Conclusion: Dephosphorylation via PP2A maintains Mcl-1 expression. Significance: Mcl-1 overexpression in chemoresistant cancer cells can be dramatically reduced by inhibiting its dephosphorylation. Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells.


Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in
Rapid up-and down-regulation of the Bcl-2 family member Mcl-1, via environmental signals, is an important determinant of viability in normal cells (1)(2)(3). However, Mcl-1 is expressed abundantly and/or in a sustained fashion in many different types of cancer (e.g. leukemias, lymphomas, and solid tumors) and renders tumor cells resistant to multiple chemotherapeutic agents (1, 2, 4 -9). Approaches to inhibit or down-regulate Mcl-1 are therefore being pursued actively (2, 6, 10 -16).
BL41-3 Burkitt lymphoma cells exhibit characteristics seen in human cancer, in particular, amplification of endogenous Mcl-1 and resistance to multiple chemotherapeutic agents (4,27,33,34). In addition, Mcl-1 degradation in these cells is not mediated via the glycogen synthase kinase-3-targeted pathway (33). Finally, ERK activation, which is also common in cancer, can be mimicked by application of the phorbol ester, 12-Otetradecanoylphorbol-13-acetate (TPA). 2 TPA markedly stabilizes Mcl-1 and exacerbates resistance in BL41-3 cells concomitantly exposed to chemotherapeutic agents. * This work was supported by National Institutes of Health Grant The present work focused on Mcl-1 dephosphorylation, which has not been studied extensively and might also be expected to influence expression. Previous studies had shown that phosphorylation is present only at very low basal levels in BL41-3 cells, and is increased in the presence of the phosphatase inhibitor OA (26,27). A possible explanation for this was that Mcl-1 was subject to dephosphorylation, which was inhibited by OA. This possibility was examined here, using antibodies that specifically recognized phosphorylated Thr-163 and Ser-159. A rapid increase in phosphorylation at these sites was seen with OA, as well as with calyculin A, another agent that inhibits. Increased phosphorylation was accompanied by a precipitous decline in Mcl-1 expression, followed by cell death. Importantly, these events were maintained in cells concomitantly exposed to TPA plus OA. Mcl-1 was found to interact with PP2A in a transfectable CHO cell system, and shRNAmediated knockdown of PP2A/A␣ recapitulated the increase in Thr-163/Ser-159 phosphorylation and decrease in Mcl-1 expression. In sum, inhibition of PP2A maintains Mcl-1 phosphorylation at Thr-163/Ser-159 and dramatically reduces abundant/stabilized expression of this prosurvival protein in chemoresistant cancer cells.
BL41-3 cells were resuspended in fresh medium 1 day before exposure to the phosphatase inhibitors okadaic acid, calyculin A, and tautomycin (EMD Millipore) (38 -40). Because effects on a marker of PP2A inhibition (phospho-ERK (41, 42)) were not observed with fostriecin (1 nM to 1 M, from EMD Millipore or Alexis Biochemicals; see REf. 43), this phosphatase inhibitor was not further pursued. U0126 was from EMD Millipore, and LiCl was from Sigma.
Constructs-The WT Mcl-1 and phosphomutant constructs (in pcDNA3.1) have been described (26,33) as has the PP2A/A␣ shRNA construct (in pcDNA3.1/H1-TO) (44). The control shRNA is identical to the latter except that PP2A/A␣ shRNA is replaced by sequence not represented in the human genome (insert: GATCCCCTTCGAAGCTCGCTATCTGCT-TCAAGAGAGCAGATAGCGAGCTTCGAATTTTTGG-AAA, where the replacement sequence is underlined). The hemagglutinin (HA)-tagged PP2A/C construct (45) and the GST-Mcl-1 and GST constructs (21) have been described. The latter two were expressed in SRP Escherichia coli grown in LB medium at 30°C as described (46,47).
Antibodies and Western Analysis-Ser-159 phosphorylation was monitored using a polyclonal antibody (Cell Signaling Technology, catalog no. 4579), which was found to recognize phosphorylation at Ser-159 but not Thr-163; although the antibody was originally directed against both these sites, the signal was essentially completely lost in the presence of a T159A mutation (data not shown). Different lots of the antibody exhibited stronger versus weaker phospho-Ser-159 signal. A previously described phospho-Thr-163-directed antibody was used in initial experiments (33) as indicated in the figure legends. Other figures utilized an antibody being developed by Cell Signaling Technology (no. BL13917 (3131)), which became available when the supply of the initial antibody was exhausted and which found to yield improved detection of pThr-163 Mcl-1. Antibodies recognizing Mcl-1 (S-19) and HA (F-7) were purchased from Santa Cruz Biotechnology. Rabbit antibodies directed against GAPDH, PP2A A subunit, PP2A C subunit, phospho-p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), ␤catenin, and poly(ADP-ribose) polymerase (PARP) were from Cell Signaling Technology.
Western blotting was performed as described (33). Blots were imaged using the ChemiDoc Molecular Imaging System (Bio-Rad), and band density was quantitated using ImageJ (NIH). SigmaStat (Systat Software, Inc.) and Prism (GraphPad Software, Inc.) were used for statistical analysis.
Co-immunoprecipitation and GST Pulldown-CHO cells were co-transfected with WT Mcl-1 and HA PP2A/C and, after 24 -48 h, cells were washed in ice-cold PBS followed by lysis in CHAPS buffer (20 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% CHAPS, 10 mM NaF, 20 mM Na 3 PO 4 , and 5 mM Na 2 HPO 4 ) to which 1 mM DTT and protease inhibitor mixture (Sigma) were added just before use. After mixing for 15 s and centrifugation at 16,000 ϫ g for 10 min, the supernatant was incubated (4 h at 4°C with rotation) with an antibody directed against either Mcl-1 or HA, in the presence of protein A/G beads (Sigma). After centrifugation at 2039 ϫ g for 2 min, the beads were either washed twice with ice-cold CHAPS buffer containing DTT and protease inhibitors or passed twice through a 1 M sucrose cushion via centrifugation. Following washes, the beads were suspended in Laemmli buffer and boiled, and Western blotting was carried out.
Mcl-1 GST protein immobilized on glutathione-agarose beads (Sigma) was tested for the ability to pulldown endogenous PP2A from BL41-3 or CHO cell lysates, where an approximately equivalent amount of GST served as a control. For this purpose, cells were washed 1ϫ in PBS and resuspended in 20 mM Tris buffer containing 12.5% glycerol, 0.2% Nonidet P-40, 200 mM NaCl, 1 mM EDTA, and 1 mM EGTA (48), to which 1 mM DTT and protease inhibitor mixture were added as above. After lysis by vortexing 3ϫ (1-min each), followed by centrifugation at 16,000 ϫ g for 10 min, the supernatant was incubated with the beads for 4 h at 4°C. The beads were then washed three times in ice-cold PBS, followed by resuspension in Laemmli buffer and boiling. Expression of the GST proteins was monitored by Coomassie Blue staining of SDS-PAGE gels, and PP2A was detected by Western blot analysis.
Cell Death Assays-Previously described methods were used to assess PARP cleavage and to examine cell morphology using stained cytospin slide preparations (33). Slides were examined independently by two board-certified pathologists in a blind fashion. The CellTox Green Cytotoxicity assay was performed as suggested by the manufacturer (Promega).

Okadaic Acid Results in a Rapid Increase in Mcl-1 Phosphorylation at Thr-163 and Ser-159, along with a Decrease in Expression in
Mcl-1-amplified Lymphoma Cells-In previous studies, an increase 32 P-radiolabeling of Mcl-1 had been observed in BL41-3 cells exposed to 1 M OA (27), a concentration that preferentially inhibits PP2A in other intact cells (49). A range of concentrations was therefore tested with a 6 h-exposure: phosphorylation at Thr-163 and Ser-159 was increased, most prominently at 0.25 M OA (Fig. 1A, lane 3).
Mcl-1 expression was decreased at this and higher concentrations, which was perhaps not unexpected given that these sites lie in a phosphodegron (22)(23)(24)29). Phospho-ERK was monitored in parallel, because ERK is a substrate of PP2A (41,42). Accordingly, phospho-ERK, while essentially undetectable in untreated cells, was readily detectable in cells treated with OA ( Fig. 1A, bottom panel, and see below). In sum, OA increased this marker of PP2A inhibition and also increased phosphory-lation at Thr-163/Ser-159 while decreasing expression of Mcl-1. This initial observation set the stage for further exploring the possibility that the PP2A inhibitor OA prevents Mcl-1 dephosphorylation and causes a decline in its expression.
Time course studies showed that the effects of OA occurred rapidly, increased phosphorylation being seen within ϳ1-3 h (Fig. 1B, lanes 2 and 3). Increased Ser-159 phosphorylation and decreased Mcl-1 expression became apparent together, as shown in the upper and lower graphs in Fig. 1C where different symbols represent independent experiments. After reaching a peak, the Ser-159 phosphorylated species declined in tandem with total Mcl-1 protein (Fig. 1B, lanes 3-5, and Fig. 1C). Thr-163 phosphorylation in initial experiments was apparent 1 h after the application of OA, which was before the peak in Ser-159 phosphorylation (Fig. 1B, lanes 2 and 3). The use of closely spaced time points confirmed that Thr-163 phosphorylation was prominent at 1 h and was transient (Fig. 1D). The timing of these events probably reflects the fact that Thr-163 phosphorylation primes for Ser-159 phosphorylation ((23) and comparable data in Mcl-1-transfected CHO cells, data not shown) and Ser-159 phosphorylation can target Mcl-1 for degradation (22,24,29). Overall, these findings were consistent with the premise that Mcl-1 undergoes dephosphorylation in untreated BL41-3 cells (resulting in barely detectable basal phosphorylation at the Thr-163/Ser-159 phosphodegron) and that dephosphorylation is prevented by the PP2A inhibitor OA.

Mcl-1 Phosphorylation Is Increased, and Expression Decreased, in the Presence of Calyculin A but Not Tautomycin-
Given the striking effects observed with OA, two additional phosphatase inhibitors were tested: calyculin A (CA), which inhibits PP2A and PP1 non-selectively, and tautomycin, which has lower potency for inhibition of PP2A than for inhibition of PP1 (40,50). In time course studies, CA had effects remarkably similar to those seen with OA, increasing the phospho-ERK marker and increasing Mcl-1 phosphorylation while decreasing its expression ( Fig. 2A). The effects of various concentrations of OA or CA versus tautomycin were therefore examined, initially at a 1-h time point before extensive loss of Mcl-1. Although phosphorylation was potently increased with OA (Ն0.25 M) or CA (Ն0.01 M; Fig. 2B, lanes 1-10), only a minimal increase was seen with tautomycin at the highest concentration tested (1 M) (Fig. 2B, lanes 11-15). Upon examination at a 3-h time point, Mcl-1 expression was markedly decreased with OA or CA even at the lowest concentrations tested (Fig. 2C, lanes 1-5, and see legend), whereas tautomycin had little effect except at the 1 M concentration, which also increased phospho-ERK (Fig. 2C, lanes 6 -10). The profile of these effects implicated PP2A inhibitors in increasing phosphorylation at Thr-163 and Ser-159 and decreasing expression of Mcl-1.
Phosphorylation at Thr-163 and Ser-159 with Okadaic Acid or Calyculin A Is Maintained upon Inhibition of ERK Activation by U0126, whereas Ser-159 Phosphorylation Is Inhibited by LiCl-Interesting differences were noted above in the effects of phosphatase inhibitors on Mcl-1 versus ERK. Increased phosphorylation at Thr-163 and Ser-159 was prominent at OA concentrations that produced submaximal ERK activation (Fig.  1A). In addition, effects on Mcl-1 phosphorylation were underway before the major increase in phospho-ERK (Figs. 1B and 2A). Thus, although the increase in Mcl-1 phosphorylation appeared likely to relate to PP2A inhibition (Fig. 2), it did not seem to relate to the associated ERK activation. This was further examined using U0126, at a concentration found to inhibit ERK activation with OA as expected (33). LiCl was tested in parallel, at a concentration that increases expression of the glycogen synthase kinase-3 target, ␤-catenin (33). In cells exposed to OA for 3 h, Ser-159 phosphorylation was not affected by U0126 but was inhibited by LiCl (Fig. 3A, lanes  5 and 6). This suggested the involvement of glycogen synthase kinase-3 but not ERK. In cells exposed to OA for 1 h, the Thr-163 phosphorylation prominent at this time (Fig. 1D) was not blocked in the presence of U0126 to inhibit ERK activation (Fig.  3, B and C). Similar results were seen in the case of CA, where phosphorylation was not prevented by U0126 but Ser-159 phosphorylation was inhibited by LiCl (Fig. 3D).
The U0126 insensitivity of Mcl-1 phosphorylation in the presence of OA or CA recalled earlier findings showing that the low basal Mcl-1 phosphorylation present in untreated cells is U0126-insensitive (26,27). Taken together, these results suggest that Mcl-1 undergoes basal ERK-independent phosphorylation countered by dephosphorylation, where inhibition of the latter by OA or CA maintains basal Thr-163 phosphorylation and allows LiCl-inhibitable Ser-159 phosphorylation.
Protein Phosphatase 2A Interacts with Mcl-1-In view of the above results with pharmacological inhibitors affecting PP2A (Figs. 1 and 2), the transfectable CHO cell system was used to test for an interaction between Mcl-1 and this phosphatase. The active PP2A holoenzyme consists of a scaffolding subunit (the A subunit or PP2A/A), as well as a catalytic subunit  (PP2A/C) and one of a variety of regulatory subunits (PP2A/B) (38,(51)(52)(53). In cells co-transfected with WT-Mcl-1 and HA-PP2A/C, HA-PP2A/C was co-immunoprecipitated upon immunoprecipitation of Mcl-1 (Fig. 4A). Similarly, Mcl-1 was co-immunoprecipitated with HA-PP2A/C (Fig. 4B). Co-immunoprecipitation of the endogenous PP2A/A subunit was detectable in the latter experiment although not the former (Fig. 4B, lowest blot). This is likely because the endogenous PP2A/A subunit is expressed at levels near the limit of detection (Fig. 4B,  lane 4) and/or because PP2A generally interacts transiently with substrates. Further assessment using GST pulldown demonstrated a specific interaction between GST-Mcl-1 and endogenous PP2A/A and PP2A/C, as assessed using either BL41-3 or CHO cells (Fig. 4C). In brief, GST pulldown as well as immunoprecipitation demonstrated an association between Mcl-1 and PP2A.

Knockdown of Protein Phosphatase 2A/A␣ Increases Phosphorylation at Thr-163 and Ser-159 while Decreasing Mcl-1
Expression-The interaction observed above provided an additional impetus for carrying out knockdown experiments with an shRNA that effectively targets PP2A/A␣, the major isoform of the scaffolding subunit ( Fig. 5A and legend) (44). The A subunit provides a structural base for the assembly of the PP2A holoenzyme (39), and its knockdown inhibits formation of the active complex and reduces the dephosphorylation of substrates (42,44). Co-transfection with this shRNA and WT-Mcl-1 resulted in an increase in phosphorylation at Thr-163 and Ser-159 and a decrease in Mcl-1 expression (Fig. 5). Phospho-ERK is detectable basally in CHO cells unlike in BL41-3 cells and did not exhibit a significant further increase in the presence of the shRNA. This could relate to the fact that only a proportion of cells undergoes transfection and/or to a difference from BL41-3 cells in PP2A subunit composition (42,54,55). Whatever the case, knockdown experiments in transfected CHO cells reinforced observations with pharma- and/or HA PP2A/C (1:3 (w/w)), where empty vector was added with single constructs to equalize the total amount of DNA. After 24 h, immunoprecipitation (IP) was carried out for Mcl-1, followed by Western blotting as indicated. B, CHO cells were co-transfected as above with WT-Mcl-1 and/or HA-PP2A/C (3:1 (w/w)), and immunoprecipitation was carried out with anti-HA. C, GST-Mcl-1 and GST proteins were tested for the ability to pulldown PP2A/C and PP2A/A from BL41-3 and CHO cell lysates. Below the Western blot for these proteins is the Coomassie Blue-stained SDS-PAGE gel showing the GST proteins used for pulldown. The electrophoretic migration of PP2A/C reflects the fact that this protein undergoes disulfide bridge formation in the presence of DTT resulting in a higher molecular weight band (input samples in third and sixth lanes on the upper Western blot), which reverts to the unmodified form when the concentration of DTT is reduced (first and fourth lanes) (68). Multiple bands can also occur with PP2A due to oxidation-mediated modifications as described (68 -70). cologic inhibitors of PP2A in endogenously expressing BL41-3 cells, demonstrating increased Mcl-1 phosphorylation and decreased expression.

Increased Mcl-1 Phosphorylation and Decreased Expression with Okadaic Acid Appear before Markers of Cell Death-The decrease in
Mcl-1 expression seen in BL41-3 cells exposed to OA could be an early event in cell death, as is often the case with Mcl-1 (56,57) and was noted to occur before the loss of phospho-ERK (Fig. 1C). Indeed, changes in Mcl-1 phosphorylation and expression were found to precede the appearance of a variety of markers of cell death. Although increased phosphorylation and decreased Mcl-1 expression were seen 1 h after the application of OA, PARP cleavage did not come into evidence until ϳ4 h (Fig. 6A). Similarly, morphological examination did not reveal a noticeable change at 1 h, the majority of cells appearing viable in the absence or presence of OA (Fig. 6B,  upper panels). However, at 3 h, when Mcl-1 expression had declined extensively, cells exhibited premature chromatin condensation and cell death as is typical with phosphatase inhibitors (Fig. 6B, lower panels) (58). Loss of membrane integrity became apparent at ϳ12 h (Fig. 6C). In sum, the effects of OA on Mcl-1 phosphorylation and expression were apparent within 1 h, whereas PARP cleavage and morphological death were not seen until ϳ3-4 h, and membrane integrity was lost thereafter. In agreement with findings above suggesting that the effects of OA on Mcl-1 result from phosphatase inhibition, these effects preceded the appearance of markers of cell death rather than occurring as a result of this process.
Okadaic Acid Results in a Rapid Decline in Mcl-1 Expression in the Presence as well as the Absence of TPA-The effects seen with OA provided an interesting counterpoint to previous observations with TPA, which does not cause a decline in Mcl-1 expression (26,27,33). In fact, TPA markedly slows the decline in Mcl-1 expression that occurs in the presence of chemotherapeutic agents (33). BL41-3 cells were therefore exposed to OA and TPA separately and together. Thr-163 phosphorylation at 1 h was similar with the two agents (Fig. 7A), the difference being that this increase was transient with OA ( Fig. 1D) but is sustained with TPA (for Ͼ 6 h) (33). In addition, the Mcl-1 pS159/Mcl-1 ratio was increased to a greater extent with OA than with TPA (Fig. 7B), consistent with inhibition of Mcl-1 dephosphorylation by OA in conjunction with declining expression. Importantly, rapid Mcl-1 decay and ensuing death were maintained when OA was applied together with TPA (Fig.  7, B-D). This differed from the inhibition of Mcl-1 decay and reduced cell death seen when chemotherapeutic agents are applied with TPA (33).

DISCUSSION
Mcl-1 is exquisitely controlled via up-regulation/stabilization and down-regulation/turnover, where expression level determines its effects on cell viability (1,2,8,59). Turnover of the protein can occur rapidly and is regulated by phosphorylation as well as other post-translational modifications (2,19,26,33,60). As shown by the present studies, Mcl-1 dephosphorylation also plays a key role. Thus, pharmacologic inhibitors of PP2A such as OA resulted in increased phosphorylation at Thr-  163/Ser-159 along with decreased Mcl-1 expression, and similar effects were seen in knockdown experiments. Increased Ser-159 phosphorylation in BL41-3 cells occurred in tandem with decreased expression, and these events preceded the appearance of a variety of cell death markers. The effects of OA were maintained upon concomitant application of TPA, providing a contrast to the slowed Mcl-1 decay and exacerbation of resistance seen when different types of chemotherapeutic agents are applied with TPA (33). In sum, inhibition of Mcl-1 dephosphorylation by inhibition of PP2A was capable of overriding abundant expression and stabilization of this prosurvival protein in chemoresistant cancer cells.
Although TPA activates ERK, which phosphorylates Thr-163 (26,27), OA appeared to maintain the basal ERK-independent Thr-163 phosphorylation that occurs in BL41-3 cells. ERK activation with TPA is sustained and Thr-163 phosphorylation is as well, likely because ongoing phosphorylation can counter dephosphorylation. By instead inhibiting effects of the phosphatase PP2A on Mcl-1, OA is speculated to allow coupling to subsequent steps in the degradation process to occur before dephosphorylation. Because dephosphorylation is downstream of the stimulation of phosphorylation by TPA, the effect of OA is maintained upon combined application with TPA.
Many questions remain to be addressed regarding Mcl-1 dephosphorylation. The rapidity of the effects seen with OA or CA, the interaction of Mcl-1 with the core subunits of PP2A, and the fact that effects were seen upon PP2A knockdown as well as pharmacologic inhibition, are compatible with direct dephosphorylation of Mcl-1 by PP2A. At the same time, given the complexity of the interactions between PP2A and a variety of signaling pathways, other actions of PP2A could have a role (41,42,55,(61)(62)(63). Another point is that, because of the hierarchical relationship between phosphorylation at Thr-163 and Ser-159, effects at these two sites could not be separated completely. The effect of phosphatase inhibitors on Thr-163 phosphorylation could be seen in isolation from Ser-159 phosphorylation (i.e. in the presence of LiCl). Conversely, however, loss of Thr-163 phosphorylation itself prevents Ser-159 phosphorylation (i.e. an T163A as well as an S159A mutation result in loss of Ser-159 phosphorylation (23) and data not shown). Therefore, the effect of phosphatase inhibition on Ser-159 phosphorylation could not be examined in the absence of Thr-163 phosphorylation. A third point is that Mcl-1 is subject to phosphorylation at multiple additional sites (19,60), which are likely also affected by phosphatases.
Another area for future investigation relates to the death that follows the decline in Mcl-1 in OA-treated BL41-3 cells. Additional actions of OA that contribute to this outcome are of interest as potential targets. It is also noted that some tumors exhibit alterations in various PP2A subunits (64 -67), and it will be important to determine how this affects sensitivity to phosphatase inhibition. Overall, future studies of Mcl-1 dephosphorylation via PP2A may lead to approaches for reducing elevated/ stabilized Mcl-1 expression in cancer cells, identifying PP2A profiles that render cells sensitive to this effect and promoting death even in the presence of extensive resistance to chemotherapeutic agents.