Negative regulation of Pim-1 protein kinase levels by the B56β subunit of PP2A

Abstract

The Pim protein kinases are serine threonine protein kinases that regulate important cellular signaling pathway molecules, and enhance the ability of c-Myc to induce lymphomas. We demonstrate that a cascade of events controls the cellular levels of Pim. We find that overexpression of the protein phosphatase (PP) 2A catalytic subunit decreases the activity and protein levels of Pim-1. This effect is reversed by the application of okadaic acid, an inhibitor of PP2A, and is blocked by SV40 small T antigen that is known to disrupt B subunit binding to PP2A A and C subunits. Pim-1 can coimmunoprecipitate with the PP2A regulatory B subunit, B56β, but not B56α, γ, δ, ɛ or B55α. Using short hairpin RNA targeted at B56β, we demonstrate that decreasing the level of B56β increases the half-life of Pim-1 from 0.7 to 2.8 h, and decreases the ubiquitinylation level of Pim-1. We also find that Pin1, a prolyl-isomerase, is capable of binding Pim-1 and leads to a decrease in the protein level of Pim-1. On the basis of these observations, we hypothesize that phosphorylated Pim-1 binds Pin1 allowing the interaction of PP2A through B56β. Dephosphorylation of Pim-1 then allows for ubiquitinylation and protein degradation of Pim-1.

Introduction

The Pim family of serine (Ser)/threonine (Thr) protein kinases were initially identified as a target for proviral activation by Moloney murine leukemia virus (Cuypers et al., 1984; Selten et al., 1985). The Pim proteins have been implicated in the control of tumorigenesis, cell cycle progression and apoptosis (Wang et al., 2001; Mikkers et al., 2004; Amaravadi and Thompson, 2005; Bachmann and Moroy, 2005). In animal models, Pim protein kinases have been shown to enhance development of lymphoma induced by c-Myc, and to be overexpressed in hepatocellular cancer (Cuypers et al., 1984; Selten et al., 1985; van Lohuizen et al., 1989). In humans, the protein levels of Pim have been shown to be elevated in lymphomas (Ionov et al., 2003), leukemias (Adam et al., 2006) and prostate cancer (Cibull et al., 2006; Xu et al., 2005). It is thought that Pim inhibits apoptosis by phosphorylating the BAD protein and enhancing the activity of Bcl-2 (Aho et al., 2004; Kim et al., 2006; Macdonald et al., 2006). Alternatively, Pim has been shown to regulate nuclear factor-kappa B (NF-κB) activity and thus downstream proteins that play a role in apoptosis, that is, Bax (Hammerman et al., 2004).

Because overexpression of Pim may contribute to tumorigenesis, the mechanism by which the levels of Pim proteins are controlled is important. Both Pim-1 and -2 proteins levels are elevated transcriptionally by the application of interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, IL-7, and other cytokines to cells (Wang et al., 2001). Mitogen stimulation can regulate the stability of Pim mRNA (Fox et al., 2003). Once increased, Pim protein kinases have a relatively short half-life (about 10 min). This suggests that the degradation of Pim proteins may play a crucial role in regulating its half-life. Recently, the protein phosphatase (PP) 2A (Losman et al., 2003) and ubiquitinylation (Shay et al., 2005) have been suggested to play a role in regulating the degradation of Pim kinases.

PP2A is a heterotrimeric protein with an A subunit that serves as a scaffold binding the catalytic subunit (C) and the regulatory subunit (B). PP2A has been shown to regulate varied cellular properties including proliferation, growth, differentiation and apoptosis (Janssens and Goris, 2001; Lechward et al., 2001; Arroyo and Hahn, 2005; Janssens et al., 2005). A critical role for PP2A in transformation has been suggested by the observation that SV-40 small T (ST) antigen, a specific inhibitor of PP2A holoenzyme formation, is required for the transformation of human cells along with SV-40 large T, human telomerase reverse transcriptase, and H-Ras. Elevation of the PP2A B56γ3 subunit reverses this effect and blocks transformation (Chen et al., 2004). A further suggestion for the important role of PP2A in human neoplasia comes from the observation that human cancers, for example, breast cancer, harbor mutations in the A subunit that prevent binding of either the B or C subunit and thus inhibit PP2A activity (Ruediger et al., 2001a, 2001b).

The ability of PP2A to modulate the dephosphorylation of c-Myc, and thus control its degradation depends on the binding and regulation of c-Myc by the Pin1 prolyl-isomerase. Isomerization of proline (Pro) residues by this protein has been shown to promote dephosphorylation of various molecules by PP2A (Zhou et al., 2000; Yeh et al., 2004; Monje et al., 2005). Pin1 binds Ser/Thr-Pro motifs through its WW motif and binds to a diverse set of substrates, including Cdc25 and Tau (Zhou et al., 2000; Stukenberg and Kirschner, 2001). Through these mechanisms, Pin1 appears to play a significant role in cellular transformation.

Here, we report that PP2A regulates the level of Pim-1. Of the many PP2A B subunits, the PP2A B56β subunit appears to bind strongly to Pim-1, and knockdown of B56β increases the levels of Pim-1. In addition, Pin1 is found in a complex with Pim-1, and knockdown of Pin1 decreases the Pim-1 expression levels. These data suggest a complex regulation of Pim-1 protein levels that could be targeted during transformation.

Results

Increased PP2A activity negatively regulates Pim-1 protein levels

As shown in Figure 1a, increasing amounts of PP2A-HA-C reduced both Pim-1 and Pim-2 protein levels. As a negative control, the PP2A-HA-C did not reduce the protein levels of the transcription factor, Runx1. To demonstrate that PP2A-HA-C regulates not only transfected Pim-1 levels but the endogenous Pim-1 protein levels, we have used the murine hematopoietic cell line BaF3, which contains a measurable level of endogenous Pim-1 protein (Kim et al., 2005). Figure 1b shows that increasing cellular PP2A activity via transfection can decrease endogenous 33 and 44 kDa Pim-1 protein levels. Okadaic acid (OA) is a strong inhibitor to PP2A with lesser activity against PP1 (Boudreau and Hoskin, 2005). Treatment of Pim-1 transfected 293 T cells or BaF3 with OA dramatically increased the level of either transfected or endogenous Pim-1 (Figure 1c and d). SV40 ST can compete with the PP2A B55α subunit and some B56 subunits for binding to the PP2A-A and -C subunits and thus inhibit PP2A activity (Chen et al., 2004; Arroyo and Hahn, 2005). Figure 1e demonstrates that ST can block the PP2A-mediated reduction in Pim-1 protein levels only when PP2Ac is part of the PP2A holoenzyme. These experiments demonstrate that Pim-1 and -2 protein levels are negatively regulated by the activity of PP2A.

Figure 1
figure1

Pim-1 protein levels are negatively regulated by PP2Ac. (a) 293 T cells were co-transfected with pcDNA3/ Pim-1 or Pim-2, Runx1, and increasing amounts of pD30-PP2A-HA-C. Control empty pcDNA3 vector was added to ensure that the total amount of plasmid DNA per transfection was identical. Cell lysates were collected at 36 h post-transfection. Western blotting was carried out using HA (for exogenous PP2Ac), PP2Ac (for total PP2Ac), Pim-1, Pim2, His and GAPDH antibodies. (b) BaF3 cells were transfected with increasing amounts of pD30-PP2A-HA-C. Endog.Pim-1 Western blotting demonstrates the two murine endogenous Pim-1 proteins at 44 and 33 kDa. (c) 293 T cells were transfected with 0.1 μg pcDNA3/Pim-1. At 1 h before harvesting, transfected cells were either treated with ethanol, or increasing amounts of OA as indicated. (d) BaF3 cells were either treated with ethanol, or increasing amounts of OA as indicated for 1 h. (e) 293 T cells were co-transfected with pcDNA3/Pim-1, with or without pCEP-SV40 ST antigen, plus increasing amounts of pD30-PP2A-HA-C.

PP2A dephosphorylates Pim-1 and decreases Pim-1 kinase activity

To determine whether PP2A can dephosphorylate Pim-1 kinase a cDNA encoding Pim-1 or kinase-dead Pim-1 was transfected into 293 T cells which were subsequently labeled with [32P] orthophosphate. The Pim-1 protein kinase was immunoprecipitated by using anti-Flag beads, and then the immunoprecipitates were incubated in vitro with or without recombinant PP2A (A/C dimer). This in vitro dephosphorylation assay demonstrates that recombinant PP2A can directly dephosphorylate Pim-1 (Figure 2a, upper row, lanes 2, 3 with lane 1). The total amount of Pim-1 immunoprecipitated was identical in all four lanes (Figure 2a, the second row). To examine whether the phosphorylation of Pim-1 regulates its activity, the recombinant PP2A-treated Pim-1 immunoprecipitates bound to Flag beads were first washed to remove all PP2A and then treated with OA (100 nM) for 30 min to eliminate the effect of remaining PP2A. We also used Western blots to investigate the recombinant PP2Ac protein level remaining in the Flag beads before and after phosphate-buffer saline (PBS) washing. Figure 2b demonstrates that after washing, the PP2Ac was not detectable on the beads (compare lane 2 with 1). Following washing, the Flag beads were subjected to a kinase assay using histone H1 as a substrate. Figure 2a (the third row) demonstrates that after treating with PP2Ac, the kinase activity of Pim-1 was decreased when compared with the untreated Pim-1. The kinase-dead Pim-1 cannot phosphorylate histone H1 (Figure 2a, third row, lane 4).

Figure 2
figure2

PP2A dephosphorylates Pim-1 in vitro, and decreases Pim-1 kinase activity. (a) 293 T cells were transfected with 2 μg pcDNA3/Flag-Pim-1 (WT or Kinase-dead). After 36 h transfection, the Flag-Pim-1 proteins were labeled with [32P] orthophosphate for 4 h followed by the immunoprecipitation of Flag-Pim-1 proteins. The immunoprecipitates were treated with or without recombinant PP2A (A/C dimer) in an in vitro phosphatase reactions. (b) After incubation of PP2A/C with Pim-1, Flag beads were washed extensively with PBS to eliminate the recombinant PP2A. Western blotting was used to compare the PP2Ac level in the Flag beads before (lane 1) and after (lane 2) washing.

PP2A B56β associates with Pim-1 in vivo

We examined the ability of Pim-1 to coimmunoprecipitate with B55α and B56 family subunits. We did not find any interaction with B55α (data not shown). As shown in Figure 3a, Pim-1 coimmunoprecipitated with HA antibody only in the presence of the B56β subunit (Figure 3a, upper panel). Alternatively, when Pim-1 is immunoprecipitated with anti-Flag beads, only the B56β subunit is co-immunoprecipitated with Pim-1 (Figure 3b, upper panel). This suggests that the PP2A holoenzyme associates with Pim-1 through the B56β regulatory subunit. We also immunoprecipitated endogenous Pim-1 from BaF3 cells demonstrating association with the endogenous B56β and PP2A-C subunit occurs in non-transfected cells as well (Figure 3c).

Figure 3
figure3

Pim-1 specifically associates with B56β in vivo. (a) Pim-1 was co-transfected with 0.5 μg HA tagged B56α, -β, -γ, -δ or -ɛ subunits into 293 T cells. Whole-cell lysates were immunoprecipitated with anti-HA beads. (b) The experiment was carried out similarly to Figure 3a except that the cell lysates were immunoprecipitated with anti-Flag beads. (c) BaF3 cell lysates were immunoprecipitated with Pim-1 antibody or control IgG.

To identify the binding site of B56β on the Pim-1 protein, we prepared deletion mutants of Pim-1, as indicated in Figure 4a. The HA-tagged B56β was expressed in 293 T cells along with the Flag-tagged full length- or deleted Pim-1 mutants. Figure 4b demonstrates B56β coimmunprecipitates with full length (1–313), ΔN (69–313), ΔC (1–250) and ΔC (1–177) Pim-1, but not with ΔN (140–313). On the basis of these results, we narrowed the interaction site for Pim-1 and the B56β subunit to between amino acid residues 70 and 139 of Pim-1. This sequence contains the hinge region (121–126) of the Pim-1 kinase, which includes the ATP-binding pocket (Qian et al., 2005a).

Figure 4
figure4

Identification of Pim-1 domain responsible for banding to B56β. (a) Structural domains of Pim-1 and deletion mutants used in these experiments are represented as black bars. (b) 293 T cells were co-transfected with the HA-B56β (full length) along with the empty cDNA (mock) or the indicated Flag-tagged Pim-1 deletion mutants and then immunoprecipitated by using anti-Flag beads.

Knockdown of B56β increases Pim-1 protein expression

Figure 5a shows that each short hairpin RNA (shRNA) of B56β subunits specifically reduced expression of its intended target by 70–90%, but did not affect expression of unintended targets. We observed that only knockdown of B56β resulted in increased Pim-1 protein levels as compared with control (Figure 5b). Furthermore, co-transfection of Pim-1 with increasing amount of either shRNA B56β or vector control demonstrates that increasing the knockdown of B56β protein, further increased Pim-1 protein levels (Figure 5c). The shRNA B56β clearly decreases the endogenous B56β level in 293 T cells (see second panel Figure 5c). Transfection of this shRNA into BaF3 cells demonstrates that decreasing the level of B56β increases the endogenous level of both the Pim-1 33 and 44 kDa isoforms (Figure 5d).

Figure 5
figure5

ShRNA knockdown of B56β increases Pim-1 protein levels. (a) 293 T cells were co-transfected with pCEP4HA-B56α, -β, -γ, -δ, or -ɛ (targets); and pSUPER-shRNA expression vector (scrambled control or targeted to B56α, -β, -γ, -δ, or -ɛ, as indicated). Cells were maintained in DMEM supplemented with 2% FBS for 72 h. (b) 293 T cells were transfected with Pim-1 and pSUPER-shRNA expression vector. (c) 293 T cells were co-transfected with pcDNA3/Pim-1, and increasing amounts of pSUPER-shRNA expression vector to evaluate Pim-1 expression. (d) BaF3 cells were transfected with increasing amounts of pSUPER-shRNA expression vector and cell extracts were analysed by Western blotting with antibodies to Pim-1 or B56β.

B56β affects the half-life and ubiquitinylation of Pim-1

To determine whether PP2A-B56β can modulate the stability of Pim-1 protein, we transfected Pim-1 and then incubated cells with cycloheximide to block further protein synthesis. As shown in Figure 6a, knockdown of B56β significantly decreased the rate of Pim-1 protein degradation and increases the half-life of Pim-1 from 0.7 to 2.8 h. This result suggests that B56β regulates Pim-1 protein levels by regulating Pim-1 protein stability.

Figure 6
figure6

Knockdown of B56β increases Pim-1 protein stability and decreases Pim-1 ubiquitinylation. (a) 293 T cells were co-transfected with 1 μg pcDNA3/Pim-1, and 4 μg shRNA expression vector for 24 h. Cell lysates were prepared at the indicated time points after 100 μg/ml cycloheximide treatment. (b) 293 T cells were co-transfected with HA-ubiquitin, Flag-Pim-1, and shRNA expression vector (scrambled control or targeted to B56β) for 48 h. Cells were then maintained in DMEM containing 1% FBS with 1 μ M Bortezomib for 6 h.

In the case of the c-Myc protein, dephosphorylation by PP2A leads to ubiquitinylation and protein degradation (Yeh et al., 2004). To evaluate whether B56β activity is important for Pim-1 ubiquitinylation, we examined the effect of B56β knockdown on Pim-1 ubiquitinylation levels. To allow us to compare levels of protein ubiquitinylation, 293 T cells transfected with Pim-1 and shRNA B56β or control were treated with the proteasome inhibitor Bortezomib (Richardson and Mitsiades, 2005) for 6 h to prevent the degradation of Pim-1 protein. Figure 6b shows that ubiquitinylation of Pim-1 is decreased upon B56β knockdown when compared with the control group, although the total levels of Pim-1 was increased upon shRNA-B56β treatment. These results revealed that the increase of Pim-1 protein levels upon knockdown of B56β is due, in part, to decreased Pim-1 protein ubiquitinylation.

The Pin1 prolyl isomerase associates with Pim-1 in vivo and facilitates Pim-1 degradation

As reported, PP2A is a conformation-sensitive protein phosphatase, preferring the trans configuration of Pro residue adjacent to the phosphor-Ser or -Thr in substrates (Zhou et al., 2000). The prolyl-isomerase Pin1 can catalyse the isomerization of Pro residues in phospho-proteins to promote their dephosphorylation by PP2A (Stukenberg and Kirschner, 2001). As shown in Figure 7a, Pim-1 can coimmunoprecipitate Pin1 when both are transfected into 293 T cells. Interestingly Pim-1 can bind and coimmunoprecipitate both Pin1 and B56β suggesting that they may form a complex in vivo. Figure 7b (upper panel) demonstrates that by using a shRNA, we can decrease the Pin1 expression approximately 70% (using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control). Conversely, knockdown of Pin1 by using shRNA led to a 2–3 fold increase in Pim-1 protein levels (Figure 7b, bottom panel). These results are consistent with the model that phospho-Pim-1 binds Pin1 leading to a configuration change that encourages the binding of PP2A with dephosphorylation of the protein leading to ubiquitinylation and degradation.

Figure 7
figure7

The Pin1 isomerase associates with Pim-1 and B56β and facilitates Pim-1 degradation. (a) 293 T cells were co-transfected with HA-Pin1, and (or) Flag-Pim-1, HA-B56β, as indicated. Anti-Flag immunoprecipitations were carried out on cleared lysates. (b) (Upper panel) 293 T cells were co-transfected with HA/Pin1, and increasing amounts of shRNA-Pin1. (bottom panel) Cells were co-transfected with pcDNA3/Pim-1, and increasing amounts of shRNA-Pin1.

Knockdown of B56β increases cell viability in Pim-positive but not in Pim-negative cell lines

To examine the effect of knocking down B56β on cell growth, we compared the effect of knockdown of B56β on two IL-3-dependent cell lines, murine BaF3 cells that have a normal level of Pim-1 and Pim-2, and bone marrow (BM) cells from Pim-1, 2 double-knockout mouse (Chen et al., 2002). Because the IL-3-dependent BM cells come from outbred mice, BaF3 cells were used as a control. As shown in Figure 8a, knockdown of B56β can increase the viability of BaF3 cells by 37% when compared with a control shRNA transfected into these cells. In contrast, transfection of shRNA B56β into the Pim knockout mouse cell line decreases this B56β subunit but has no effect on cell viability (Figure 8a). Figure 8b demonstrates that shRNA B56β can decrease the endogenous B56β level in BaF3 and BM mononuclear cells, and can increase the Pim-1 protein levels in the BaF3 cells. This result suggests that changes in the level of B56β subunit can regulate cell viability, possibly by affecting the levels of Pim proteins.

Figure 8
figure8

Knockdown of B56β affects cell viability through Pim kinase. (a) Pim-1 and Pim-2 double knockout mouse and BaF3 cells were transfected with either shRNA expression vector (scrambled control or targeted to B56β) and allowed to grow for 48 h. Data shown is the mean of three independent MTT assays. Error bars are the standard error with a student's t-test indicating a significant difference P<0.05 (*), comparing experimental with the control group. (b) Western blotting was used to measure the shRNA effect on B56β and Pim-1 expression.

Discussion

We demonstrate that in 293 T cells transfected with Pim-1 and Pim-2 cDNAs, as well as in BaF3 cells expressing endogenous levels of Pim-1, the overexpression of the PP2Ac subunit decreases the level of Pim proteins. These results extend the work of Losman et al. (2000), who have demonstrated similar findings for Pim-1 and -3. Our results demonstrate that this effect of PP2Ac is reversed by both OA, a known inhibitor of PP2Ac and expression of SV-40 ST protein (Arroyo and Hahn, 2005) and equivalent results are obtained for both Pim-1 and -2. ST is thought to displace several B55 and B56 isoforms from the trimeric PP2A forms (Van Hoof and Goris, 2004) leading to cellular transformation. This result suggests that the PP2A holoenzyme is required to decrease Pim-1 levels. By labeling cells with [32P]orthophosphate, we have been able to demonstrate that recombinant PP2A actually dephosphorylates the Pim-1 protein in vitro. The decrease in phosphorylation of Pim-1 is associated with a decreased ability of Pim-1 to phosphorylate an artificial substrate, suggesting that phosphorylation controls Pim-1 activity. Crystallography data have suggested that Pim-1 autophosphorylates and is active without an additional protein kinase cascade controlling its ability to modify substrates (Qian et al., 2005b). It is interesting that Pim-1 has been shown to transform cells in concert with both c-Myc (Verbeek et al., 1991; Shirogane et al., 1999; Ellwood-Yen et al., 2003) or Akt (Amaravadi and Thompson, 2005), and that the levels of both of these proteins are regulated by PP2A (Sears, 2004; Ugi et al., 2004; Van Kanegan et al., 2005). Using a model in which hematopoietic cells are grown in an IL-3-dependent fashion, we find that knocking down a PP2A B subunit increases viability only in Pim containing, but not in Pim knockout cells. Although it is highly likely that the B subunit could have many binding partners other than Pim, this result suggests that the B subunit by regulating Pim levels could potentially regulate cell viability.

PP2A is targeted to specific substrates through its B subunit. c-Myc interacts with the B56α subunit, and knock down of B56α can increase c-Myc protein expression levels. Dephosphorylation of Bcl-2 by PP2A through B56α appears to be required for ceramide-induced cell death (Ruvolo et al., 2002). Even in the presence of 2% NP-40, we find that the B56β subunit of PP2A interacts strongly with Pim-1. The suggestion that B56β is important for Pim-1 regulation is further strengthened by experiments in which shRNA B56β can increase the protein levels of either transfected or endogenous Pim-1, whereas other B56 subunits shRNA did not affect the Pim-1 expression. We have demonstrated that c-Myc can be conjugated to ubiquitin for ubiquitin-mediated protein degradation, and that inhibition of proteasomal degradation increases its half-life (Yeh et al., 2004). We show that decreasing the level of B56β subunit by shRNA decreases the ubiquitinylation of Pim-1 and increases the half-life of this protein from 0.7 to 2.8 h. These results suggest that the degradation of Pim-1 could be regulated in a similar fashion to c-Myc and other proteins (Janssens and Goris, 2001; Yeh et al., 2004). In this case, PP2A dephosphorylation leaves a partially phosphorylated protein, for example c-Myc with T58 phosphorylated, that is an excellent target for a specific E3 ligase. As both of Myc and Pim-1 can be negatively regulated by PP2A through different B56 subunits and play cooperative roles in lymphomagenesis, the coordinated role of PP2A on Pim-1 and Myc activity in lymphomas is worth further investigation.

We demonstrate that Pin1 can form a complex with Pim-1. Murine Pim-1 contains five potential SP docking sites for Pin1, two of these SP sites are located close together S55 and S61. The presence of two closely phosphorylated SP motifs, for example in c-Myc and RNA polymerase II, increases the binding affinity and catalytic efficacy of Pin1 towards these substrates (Xu et al., 2003; Yeh et al., 2004). This may also be true for Pim-1. Pin1 binds to phosphorylated Pim-1 and enhances the ability of the PP2A holoenzyme to bind to the complex through the B56β protein. PP2A dephosphorylates Pim-1 protein and decreases its activity. The partially phosphorylated Pim-1 is then ubiquitinylated and degraded by the proteasome (see hypothetical model Figure 9). Additional experiments will be necessary to validate completely this model and examine whether in human neoplasmas this mechanism is defective leading to increased levels of Pim protein.

Figure 9
figure9

Hypothetical model of Pim-1 degradation by the proteasome mediated by Pin1 and PP2A.

Materials and methods

Chemicals and reagents

The chemical and reagents were purchased from the listed vendors: anti-Flag M2 Agarose, protease inhibitor cocktail, N-ethylmaleimide, OA and cycloheximide (Sigma, St Louis, MO, USA); protein A/G agarose (Calbiochem, La Jolla, CA, USA); HA antibody (Abcam, Cambridge, MA, USA); GAPDH antibody (Chemicon, Temecula, CA, USA); His antibody (Qiagen, Valencia, CA, USA); Pim-1 (12H8), Pim-2 (1D12) and B56β antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); [32P]orthophosphate and [γ-32P]ATP (Perkin Elmer, Wellesley, MA, USA); recombinant PP2A, PP2Ac antibody and histone H1 (Upstate, Chicago, IL, USA); and mouse IL-3 (R&D Systems, Minneapolis, MN, USA). Bortezomib (Velcade, PS-341) was a gift of Millenium, Inc. (Cambridge, MA, USA).

Cell culture and transfection

Human embryonic kidney (HEK) 293 T cells were grown in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. Cells were plated to achieve 50–70% confluence 24 h before transfection. Transfections were performed using Effectene (Qiagen, Valencia, CA, USA) according to the instructions of the manufacturer. Transfected cells were maintained in DMEM supplemented with 10% FBS, except in shRNA experiments, in which they were maintained in 2 or 0.2% FBS for the indicated time periods. The murine hematopoietic cell line, BaF3, was cultured in RPMI 1640 medium supplemented with 10% FBS and 1 ng/ml mouse IL-3.

Plasmids

Constructs encoding pD30-PP2A-HA-C, shRNA B56 subunits have been described previously (Arnold and Sears, 2006). Expression vectors for pCEP4HA-B56α, -β, -γ, -δ and ɛ, were generous gifts from David Virshup (Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA) (McCright et al., 1996). pCEP-small-T-antigen expression vector was kindly provided by William C Hahn (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA) (Chen et al., 2004). pcDNA3/Flag-Pim-1(murine), pcDNA3/Flag-Pim-2(murine), pcDNA3/Flag-Pim-1 kinase-dead (K67A) have been described previously (Chen et al., 2005). pcDNA3/HA-Pin1 was a gift from Ilan R Kirsch (Research Oncology, Amgen, Seattle, WA, USA) (Campaner et al., 2005). HA-ubiquitin was a gift from Mathias Treier (European Molecular Biology Laboratory, Heidelberg, Germany) (Treier and Bohmann, 1994). shRNA Pin1 (TRCN0000001034) was purchased from Sigma.

Western blotting and immunoprecipitations

Western blotting was carried out as described previously (Chen et al., 2005). Primary antibodies were in 5% bovine serum albumin (BSA), 0.1% Tween20, Tris-buffered saline and were detected with HRP-conjugated secondary antibodies using the ECL reagents from Amersham (Piscataway, NJ, USA). Cleared cell lysates were incubated with indicated antibodies for 3 h followed by the addition of Protein A/G agarose for 1 h. Immunoprecipitates were washed three times with cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, pH 7.4, 1% nonylphenyl-polyethylene glycol (NP-40), 0.1% sodium dodecyl sulphate, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM ethylenediamine-N,N,N′,N′-tetraacetic acid, 1 mM sodium orthovanadate (Na3VO4), 1 mM sodium fluoride (NaF) and protease inhibitor cocktail).

Detection of ubiquitinylated Pim-1

The method has been described previously (Yeh et al., 2004). Briefly, HEK-293 T cells were co-transfected with 1 μg CMV-HA-ubiquitin, 1 μg pcDNA3/Flag-Pim-1, and 4 μg shRNA expression plasmids for 48 h. Cells were lysed with RIPA buffer, with the addition of the de-ubiquitinase inhibitor 5 mM N-ethylmaleimide (Sigma) at a cell-to-volume ratio of 1 × 106 cells/ml. Ubiquitinylated proteins were immunoprecipitated from cell lysates with an anti-Flag M2 agarose for 3 h. Immunoprecipitates were completed as described (Yeh et al., 2004).

Dephosphorylation and kinase assay

HEK-293 T cells were transfected with Flag-Pim-1 (wild-type (WT) or kinase-dead) for 36 h, then washed once and incubated with phosphate-free media containing 10% phosphate-free FBS (Invitrogen, Carlsbad, CA, USA) for 1 h. Cells were then incubated in medium containing 50 μCi/ml [32P]orthophosphate for 4 h. To immunoprecipitate Pim-1, anti-Flag M2 agarose was added to the cell lysate, followed by 3 h incubation. To study the ability of PP2A to dephosphorylate Pim, immunoprecipitates were washed once in phosphatase buffer (50 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), pH 7.5, 0.5% BSA, and 1 mM dithiothreitol (DTT), and then resuspended in 40 μl of the same buffer. Recombinant PP2A (A/C dimer) was then added and incubated at 30°C for 30 min with agitation. The reaction was terminated by washing twice in 50 mmol/l HEPES, pH 8.0. Forty percent of the reaction products were analyzed by autoradiography, 10% was taken for western blotting to measure Pim-1 levels, 50% was used in a kinase assay. First the beads were washed extensively in PBS to remove the PP2A and then treated with OA (100 nM) for 30 min to eliminate the effect of remaining PP2A. The beads were then resuspended in kinase reaction buffer (10 mM 3-morpholinopropanesulfonic acid, pH 7.4, 100 μ M ATP, 15 mM magnesium chloride, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT, and protease inhibitor cocktail, 100 nM OA). In each reaction (30 μl), 3 μg of histone H1 protein was used as substrate, and 10 μCi of [γ-32P]ATP was then added. Incubation was carried out at 30°C for 30 min with agitation.

Pim-1 half-life measurement

A total of 100-mm dishes of 293 T cells were co-transfected with 1 μg pcDNA3/Pim-1, and 4 μg shRNA expression vector (scrambled control or targeted to B56β) for 24 h. Each transfection mixture was split into six 60-mm dishes for 24 h and then starved in 0.2% FBS for 48 h. Cells were treated with 100 μg/ml cycloheximide 5 min before starting the indicated time course, and cell lysates were prepared at the indicated time points after treatment. Pim-1 protein levels were quantified relative to GAPDH levels and graphed as percent Pim-1 protein remaining.

Cell viability assay

Cells were transfected with 2 μg shRNA expression vector for 48 h. Transfected cells were counted and seeded in 96-well dishes at 1250 cells/well. After 48 h, the total cell viability was determined by a modified 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA). Metabolically active cells were measured by recording the absorbance at 490 nm using a microplate reader.

Isolation of mouse BM mononuclear cells

BM cells were flushed from the femus into RPMI 1640 containing 10% FBS and 5 ng/ml of IL-3 using a 21-gauge needle and syringe. BM cells from 3 to 5 mice were pooled and centrifuged through Histopaque 1083 (Sigma) to isolate BM mononuclear cells.

References

  1. Adam M, Pogacic V, Bendit M, Chappuis R, Nawijn MC, Duyster J et al. (2006). Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL. Cancer Res 66: 3828–3835.

  2. Aho TL, Sandholm J, Peltola KJ, Mankonen HP, Lilly M, Koskinen PJ . (2004). Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site. FEBS Lett 571: 43–49.

  3. Amaravadi R, Thompson CB . (2005). The survival kinases Akt and Pim as potential pharmacological targets. J Clin Invest 115: 2618–2624.

  4. Arnold HK, Sears RC . (2006). Protein phosphatase 2A regulatory subunit B56alpha associates with c-myc and negatively regulates c-myc accumulation. Mol Cell Biol 26: 2832–2844.

  5. Arroyo JD, Hahn WC . (2005). Involvement of PP2A in viral and cellular transformation. Oncogene 24: 7746–7755.

  6. Bachmann M, Moroy T . (2005). The serine/threonine kinase Pim-1. Int J Biochem Cell Biol 37: 726–730.

  7. Boudreau RT, Hoskin DW . (2005). The use of okadaic acid to elucidate the intracellular role(s) of protein phosphatase 2A: lessons from the mast cell model system. Int Immunopharmacol 5: 1507–1518.

  8. Campaner S, Kaldis P, Izraeli S, Kirsch IR . (2005). Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint. Mol Cell Biol 25: 6660–6672.

  9. Chen W, Possemato R, Campbell KT, Plattner CA, Pallas DC, Hahn WC . (2004). Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell 5: 127–136.

  10. Chen WW, Chan DC, Donald C, Lilly MB, Kraft AS . (2005). Pim family kinases enhance tumor growth of prostate cancer cells. Mol Cancer Res 3: 443–451.

  11. Chen XP, Losman JA, Cowan S, Donahue E, Fay S, Vuong BQ et al. (2002). Pim serine/threonine kinases regulate the stability of Socs-1 protein. Proc Natl Acad Sci USA 99: 2175–2180.

  12. Cibull TL, Jones TD, Li L, Eble JN, Ann Baldridge L, Malott SR et al. (2006). Overexpression of Pim-1 during progression of prostatic adenocarcinoma. J Clin Pathol 59: 285–288.

  13. Cuypers HT, Selten G, Quint W, Zijlstra M, Maandag ER, Boelens W et al. (1984). Murine leukemia virus-induced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region. Cell 37: 141–150.

  14. Ellwood-Yen K, Graeber TG, Wongvipat J, Iruela-Arispe ML, Zhang J, Matusik R et al. (2003). Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 4: 223–238.

  15. Fox CJ, Hammerman PS, Cinalli RM, Master SR, Chodosh LA, Thompson CB . (2003). The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes Dev 17: 1841–1854.

  16. Hammerman PS, Fox CJ, Cinalli RM, Xu A, Wagner JD, Lindsten T et al. (2004). Lymphocyte transformation by Pim-2 is dependent on nuclear factor-kappaB activation. Cancer Res 64: 8341–8348.

  17. Ionov Y, Le X, Tunquist BJ, Sweetenham J, Sachs T, Ryder J et al (2003). Pim-1 protein kinase is nuclear in Burkitt's lymphoma: nuclear localization is necessary for its biologic effects. Anticancer Res 23: 167–178.

  18. Janssens V, Goris J . (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353: 417–439.

  19. Janssens V, Goris J, Van Hoof C . (2005). PP2A: the expected tumor suppressor. Curr Opin Genet Dev 15: 34–41.

  20. Kim KT, Baird K, Ahn JY, Meltzer P, Lilly M, Levis M et al. (2005). Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival. Blood 105: 1759–1767.

  21. Kim KT, Levis M, Small D . (2006). Constitutively activated FLT3 phosphorylates BAD partially through pim-1. Br J Haematol 134: 500–509.

  22. Lechward K, Awotunde OS, Swiatek W, Muszynska G . (2001). Protein phosphatase 2A: variety of forms and diversity of functions. Acta Biochim Pol 48: 921–933.

  23. Losman JA, Chen XP, Vuong BQ, Fay S, Rothman PB . (2003). Protein phosphatase 2A regulates the stability of Pim protein kinases. J Biol Chem 278: 4800–4805.

  24. Macdonald A, Campbell DG, Toth R, McLauchlan H, Hastie CJ, Arthur JS . (2006). Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL. BMC Cell Biol 7: 1.

  25. McCright B, Rivers AM, Audlin S, Virshup DM . (1996). The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 271: 22081–22089.

  26. Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E, Jonkers J et al. (2004). Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol Cell Biol 24: 6104–6115.

  27. Monje P, Hernandez-Losa J, Lyons RJ, Castellone MD, Gutkind JS . (2005). Regulation of the transcriptional activity of c-Fos by ERK A novel role for the prolyl isomerase PIN1. J Biol Chem 280: 35081–35084.

  28. Qian KC, Studts J, Wang L, Barringer K, Kronkaitis A, Peng C et al. (2005a). Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase. Acta Crystallograph Sect F Struct Biol Cryst Commun 61: 96–99.

  29. Qian KC, Wang L, Hickey ER, Studts J, Barringer K, Peng C et al. (2005b). Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase. J Biol Chem 280: 6130–6137.

  30. Richardson PG, Mitsiades C . (2005). Bortezomib: proteasome inhibition as an effective anticancer therapy. Future Oncol 1: 161–171.

  31. Ruediger R, Pham HT, Walter G . (2001a). Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the A alpha subunit gene. Oncogene 20: 10–15.

  32. Ruediger R, Pham HT, Walter G . (2001b). Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene. Oncogene 20: 1892–1899.

  33. Ruvolo PP, Clark W, Mumby M, Gao F, May WS . (2002). A functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function. J Biol Chem 277: 22847–22852.

  34. Sears RC . (2004). The life cycle of C-myc: from synthesis to degradation. Cell Cycle 3: 1133–1137.

  35. Selten G, Cuypers HT, Berns A . (1985). Proviral activation of the putative oncogene Pim-1 in MuLV induced T-cell lymphomas. EMBO J 4: 1793–1798.

  36. Shay KP, Wang Z, Xing PX, McKenzie IF, Magnuson NS . (2005). Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway. Mol Cancer Res 3: 170–181.

  37. Shirogane T, Fukada T, Muller JM, Shima DT, Hibi M, Hirano T . (1999). Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Immunity 11: 709–719.

  38. Stukenberg PT, Kirschner MW . (2001). Pin1 acts catalytically to promote a conformational change in Cdc25. Mol Cell 7: 1071–1083.

  39. Treier M SL, Bohmann D . (1994). Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78: 787–798.

  40. Ugi S, Imamura T, Maegawa H, Egawa K, Yoshizaki T, Shi K et al. (2004). Protein phosphatase 2A negatively regulates insulin's metabolic signaling pathway by inhibiting Akt (protein kinase B) activity in 3T3-L1 adipocytes. Mol Cell Biol 24: 8778–8789.

  41. Van Hoof C, Goris J . (2004). PP2A fulfills its promises as tumor suppressor: which subunits are important? Cancer Cell 5: 105–106.

  42. Van Kanegan MJ, Adams DG, Wadzinski BE, Strack S . (2005). Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem 280: 36029–36036.

  43. Van Lohuizen M, Verbeek S, Krimpenfort P, Domen J, Saris C, Radaszkiewicz et al. (1989). Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell 56: 673–682.

  44. Verbeek S, van Lohuizen M, van der Valk M, Domen J, Kraal G, Berns A . (1991). Mice bearing the E mu-myc and E mu-pim-1 transgenes develop pre-B-cell leukemia prenatally. Mol Cell Biol 11: 1176–1179.

  45. Wang Z, Bhattacharya N, Weaver M, Petersen K, Meyer M, Gapter L et al. (2001). Pim-1: a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis. J Vet Sci 2: 167–179.

  46. Xu Y, Zhang T, Tang H, Zhang S, Liu M, Ren D et al. (2005). Overexpression of PIM-1 is a potential biomarker in prostate carcinoma. J Surg Oncol 92: 326–330.

  47. Xu YX, Hirose Y, Zhou XZ, Lu KP, Manley JL . (2003). Pin1 modulates the structure and function of human RNA polymerase II. Genes Dev 17: 2765–2776.

  48. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G et al. (2004). A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 6: 308–318.

  49. Zhou XZ, Kops O, Werner A, Lu PJ, Shen M, Stoller G et al. (2000). Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins. Mol Cell 6: 873–883.

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Acknowledgements

We thank David Virshup (University of Utah), William C Hahn (Harvard Medical School), Ilan R Kirsch (Research Oncology, Amgen, Seattle, WA, USA), Mathias Treier (European Molecular Biology Laboratory, Heidelberg, Germany) and Yusuf A Hannun (Medical University of South Carolina) for providing plasmids, Craig B Thompson (Abramson Family Cancer Research Institute, Philadelphia, PA) for providing Pim-1,2 knockout mice, Changmin Chen for growing the mouse bone marrow cells and WeiWei Chen for making the Flag-Pim-1 constructs. This work was supported by DOD Grant W81XWH-05-1-0126 to Kraft, DOD Grant W81XWH-04-1-0887 to Lilly, NIH training grant 5-T32-GM08617 and OHSU TL Tartar Trust Research Fellowship AMEDG0063 to Arnold, and NIH Grants R01-CA100855 and K01-CA086957 to Sears.

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Correspondence to A S Kraft.

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Ma, J., Arnold, H., Lilly, M. et al. Negative regulation of Pim-1 protein kinase levels by the B56β subunit of PP2A. Oncogene 26, 5145–5153 (2007). https://doi.org/10.1038/sj.onc.1210323

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Keywords

  • Pim-1
  • PP2A
  • B56β
  • protein kinase
  • Pin1

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