Carboxyl methylation of PP2A was discovered well over a decade ago, but its function remained obscure until the discovery that the addition and removal of the single methyl group on the PP2A carboxyl-terminal leucine could differentially regulate the assembly of certain PP2A heterotrimers (22
). The cloning of LCMT-1 as a mammalian PP2A methyltransferase by De Baere et al.
) has facilitated the investigation the contribution of this enzyme toward PP2A methylation and function. However, until the recent advent of small interfering RNA approaches for mammalian cells, much advancement in our understanding has come from experiments performed in yeast because of the ease of performing genetic analyses. In fact, before this study, nothing was known about the importance of LCMT-1 for normal cell cycle progression and cell survival. In this report we have utilized lentiviral shRNA and Genetrap knock-out approaches to investigate LCMT-1 function in mammalian cells. We show that a strong reduction in LCMT-1 not only results in a substantial reduction in PP2A methylation in mammalian cells but also causes a decrease in Bα
subunit association with C subunit, induces apoptosis as evidenced by caspase activation, membrane blebbing, nuclear condensation and fragmentation, and cell death, and sensitizes HeLa and HCT116 cancer cells to the microtubule targeting drug nocodazole.
Although LCMT-1 was originally purified as a PP2A methyltransferase, only a small percentage of PP2A methyltransferase activity in the original cell extract was recovered (16
), leaving open the question as to whether LCMT-1 is the major PP2A methyltransferase in mammalian cells. Our result showing that reduction of the steady-state level of PP2A C subunit methylation an average of 70% in HeLa cells and 63% in HCT116 cells by the LCMT-1 L3 shRNA clearly indicates that it is. Our observation that cells with ~10% of their normal amount of LCMT-1 protein have 30–70% of their normal amount of PP2A methylation suggests that either there is a second PP2A methyltransferase that is a minor contributor to PP2A methylation under normal growth conditions or cells have an excess of LCMT-1 for maintaining steady-state methylation levels of PP2A under cell culture conditions. The most obvious candidate for an additional PP2A methyltransferase would be the only known homolog of LCMT-1, LCMT-2. LCMT-2 was originally cloned by De Baere et al.
), who showed that it could not methylate PP2A in vitro
. Consistent with this in vitro
mammalian result, a LCMT-2 homolog exists in yeast that does not seem to contribute detectably to PP2A methylation in vivo
). These results suggest that LCMT-2 is not a PP2A methyltransferase. However, it still needs to be tested in vivo
whether LCMT-2 contributes to PP2A methylation in mammalian cells. One possible explanation for the apparent excess of LCMT-1 is that it may be necessary for dynamic responses to cellular needs or for methylation of a currently unknown additional LCMT-1 substrate. Other possible LCMT-1 substrates include the PP2A-related phosphatases PP4 and PP6. Both of these phosphatases share ~60% amino acid identity with PP2A and have identical carboxyl-terminal amino acids, including a carboxyl-terminal leucine, the known site of carboxyl methylation for both PP2A and PP4 (42
). Whether PP6 is methylated is not known. Further experiments will be necessary to determine whether LCMT-1 (or LCMT-2) methylates PP4 and/or PP6.
We have previously shown that Bα
is the PP2A B-type regulatory subunit whose assembly into heterotrimers is most dependent on PP2A C subunit methylation (24
). Our current coimmunoprecipitation results provide further support for this conclusion. LCMT-1 has been shown previously to associate with PP2A in vitro
) and to methylate PP2A on its carboxyl-terminal leucine α
carboxyl group (10
). In this study we have demonstrated further that LCMT-1 down-regulation in mammalian cells reduces both PP2A C subunit methylation and the formation of PP2ABαAC
heterotrimers and that down-regulation of these same heterotrimers by Bα
knockdown can cause part of the phenotypes seen with LCMT-1 knockdown. Moreover, we have previously shown that loss of PP2ABAC
heterotrimer formation results in selective loss of PP2A activity toward a PP2ABAC
-specific substrate (8
). Thus, it is possible that the cell death induced by LCMT-1 down-regulation may be due in part to dysregulation of Bα
heterotrimer function. A similar amount of apoptosis resulted when protein levels of either Bα
or LCMT-1 were greatly reduced. Moreover, the pancaspase inhibitor z-VAD-fmk could prevent a portion of the cell death induced by down-regulation of either LCMT-1 or Bα
, indicating that in both cases caspase-dependent and caspase-independent death pathways contribute to cell death. This similarity in the cell death observed with either LCMT-1 or Bα
reduction is again consistent with the possibility that LCMT-1 down-regulation induces death to some degree through Bα
dysregulation. However, cell death induced by down-regulation of these two proteins does not appear to be identical. It was more difficult to detect caspase activation in vivo
and in vitro
knockdown cells, and the rescue by z-VAD-fmk was consistently lower for the Bα
knockdown cells than for LCMT-1 knockdown cells. These differences suggest that LCMT-1 knockdown has other effects independent of Bα
that contribute to cell death. In yeast, deletion of the gene that encodes the LCMT-1 homolog, Ppm1p, not only reduces the level of PP2ABAC
heterotrimers 20-fold but also causes a smaller reduction (~2-fold) in PP2AB′AC
). Therefore, one possible effect unique to LCMT-1 knockdown may be reduction of PP2AB′AC
heterotrimers. Other possible targets of LCMT-1 that have yet to be tested also exist, such as PP2A heterotrimers, formed by other members (β
) of the B/B55 regulatory subunit family or by PP2A B″ regulatory subunits and PP4 and/or PP6 complexes. Although apoptosis-relevant targets of LCMT-1 other than Bα
remain to be determined, our results clearly indicate that they exist.
The increased sensitivity of both LCMT-1 and Bα
knockdown cells to the spindle-targeting drug, nocodazole, suggests that both of these proteins are important in mammalian cells for the spindle checkpoint, which ensures proper spindle formation and chromosomal attachment before progression from metaphase to anaphase. The fact that cell death was largely rescued by thymidine block-induced arrest in G1
/S indicates that cell cycle arrest, which is induced in HeLa cells by either nocodazole or thymidine, is not the sensitizing factor. Instead, the effect is specific to mitosis. This death in nocodazole- and not in thymidine-treated cells is consistent with previous results from yeast showing that loss of the Bα
homolog, Cdc55p, or of the LCMT-1 homolog, Ppm1p, causes increased sensitivity to nocodazole. However, it was not known with the increased complexity of mammalian cells whether this phenotype would be found in this system, where there are multiple members of each B-type regulatory subunit family. In mammalian cells the mechanistic details of the roles of B-type subunits in spindle checkpoint and mitotic exit are just beginning to emerge. Both the B family isoform, Bδ
, and the B′ family isoform, B′γ
, have recently been identified as negative regulators of sister chromatid separation (35
was found to protect cohesins from degradation by stabilizing securin, a negative regulator of the cohesin protease, separase. B′γ
was reported to protect cohesin degradation by binding to Shugoshin (hSgo1) and maintaining cohesins in a more stable, hypophosphorylated state. Our data suggest that Bα
is also a negative regulator of the spindle checkpoint in mammalian cells. Whether it plays yet another distinct role or shares responsibility for dephosphorylation of cohesins or securin is not known. Overexpression data such as those presented in the Bδ
/securin study do not rule out a similar role for Bα
. Likewise, B′γ
identification as an hSgo1 partner leaves open the possibility that other B-type subunits might also associate with Shugoshin proteins. Finally, a role for LCMT-1 in spindle checkpoint is consistent with a role for PP2A methylation in mitotic progression. Experiments are under way to explore this possibility further.
PP2A regulates apoptotic pathways both positively and negatively by targeting different substrates via its diverse regulatory subunits. Consequently, PP2A inhibitors and small interfering RNA down-regulation of PP2A C and A subunits have been reported to induce apoptosis or block apoptosis, depending on the system (44
). In both Drosophila
and mammalian cells, B56/B′ subunits have been reported to have an anti-apoptotic role (45
). Some evidence has also been reported in mammalian cells for an anti-apoptotic role for B″ and B subunit families (48
). In this latter study Strack et al
) presented results of four different assays analyzing the importance of B55/B family subunits for cell survival. In seeming contrast to our results, one of the assays that showed little effect was small interfering RNA knockdown of Bα
. However, the efficiency of their Bα
knockdown was not reported, making comparison to our results difficult. Moreover, in concert with our data, results from two of their other three assays supported an anti-apoptotic role for Bα
. Our finding that Bα
down-regulation induces a small amount of apoptosis that is dependent on progression through mitosis is also consistent with the ability of the adenovirus protein, E4orf4, to arrest cells in G2
/M and induce apoptosis in cancer cells in a manner dependent on its ability to bind PP2A Bα
). Our results indicate a pro-survival function for LCMT-1 as well. This function may be mediated to some extent through modulation of PP2ABαAC
heterotrimer formation, but our data suggest that Bα
-independent roles should also be considered.
The fact that knockdown of either Bα or LCMT-1 caused an enhancement of cell killing by nocodazole in HeLa cells raises the possibility that Bα heterotrimers and LCMT-1 may have potential as targets for combination chemotherapy with microtubule targeting drugs like taxanes. Importantly, both Bα and LCMT-1 knockdown HeLa cells were greatly protected from cell death by arrest in G1/S, presumably by blocking the progression of the knockdown cells into mitosis, where apoptosis was triggered due to a defective spindle checkpoint. One would expect that knockdown of these proteins would not have the same sensitizing effect to nocodazole in all cell types given that some cancer cells already have defects in checkpoints and cell cycle control. HCT116 cells also showed sensitization to nocodazole (). However, initial experiments with H1299 lung cancer cells gave a different result. Although these cells exhibit significant death when LCMT-1 is knocked down by either L2 or L3, this cell line is already more sensitive to nocodazole than HeLa or HCT116 cells and, not unexpectedly, does not show increased nocodazole sensitivity upon LCMT-1 knockdown by the L2 and L3 shRNAs (data not shown). Further experimentation will be necessary to investigate the potential of these proteins as novel drug targets.
Consistent with the observed effects of LCMT-1 reduction on cell viability, homozygous gene trap knock-out of LCMT-1 results in embryonic lethality. The expected ratio of LCMT-1+/+
progeny mice from a cross of LCMT-1+/−
mice is 1:2:1. We obtained a ratio of 1:1.4:0 () with sufficient numbers of progeny to clearly indicate that homozygous loss of LCMT-1 is lethal during development. This result could not arise simply from inviable gametes because both male and female LCMT-1+/−
mice are fertile when bred to wild-type mice.3
Based on our results with cells, this lethality could result from defects in cell cycle or increased apoptosis, but it is also possible that additional signaling defects may also contribute. To our knowledge this is the first report that LCMT-1 is essential for development. In addition, the lower than expected numbers of LCMT-1+/−
mice also raise the possibility that hemizygous loss of LCMT-1 may cause some embryonic lethality as well. Future experiments will be aimed at elucidating the nature and timing of developmental defects that lead to the death of embryos lacking this important methyltransferase enzyme.