Pim Kinases Promote Cell Cycle Progression by Phosphorylating

Research Article

Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels Daisuke Morishita,1,2 Ryohei Katayama,1 Kazuhisa Sekimizu,2 Takashi Tsuruo,1 and Naoya Fujita1

1Cancer Chemotherapy Center, Japanese Foundation for Cancer Research and 2Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Abstract kinase has three family gene products (Pim1, Pim2, and Pim3; The serine/threonine kinase Pim is known to promote cell ref. 3). They are composed of at least six translational variants: cycle progression and to inhibit apoptosis leading to Pim1 short isoform (Pim1S), Pim1 long isoform (Pim1L), Pim2 tumorigenesis. However, the precise mechanisms remain short isoform (Pim2S), Pim2 medium isoform (Pim2M), Pim2 long unclear. We show, herein, that all the Pim family members isoform (Pim2L), and Pim3 in human. Pim kinases have been (Pim1, Pim2, and Pim3) bind to and directly phosphorylate implicated in the control of tumorigenesis. In pim1 or pim2 the cyclin-dependent kinase inhibitor p27Kip1 at threonine-157 transgenic mice, Pim has been shown to enhance the development and threonine-198 residues in cells and in vitro. The Pim- of lymphoma and leukemia (2, 4–6). The expression level of Pim mediated phosphorylation induced p27Kip1 binding to 14-3-3 kinases is frequently elevated in patients with lymphoma, leukemia, protein, resulting in its nuclear export and proteasome- and prostate cancer (4, 6, 7). In particular, the expression of Pim1 has been correlated with measures of clinical outcome (8). dependent degradation. Ectopic expression of Pim kinases Kip1 Moreover, pim1 was recently identified as a target of aberrant overcome the G1 arrest mediated by wild-type p27 but not by phosphorylation-resistant T157A-p27Kip1 or T198A-p27Kip1. somatic hypermutation in non–Hodgkin’s lymphoma and B-cell Kip1 In addition to the posttranslational regulations, p27 lymphoma (9–11), and some of the mutations significantly increase promoter assay revealed that Pim kinases also had the ability Pim1 enzymatic activity (12). Thus, it is suggested that Pim kinase Kip1 to suppress p27 transcription. Pim-mediated phosphory- overexpression and activation induce tumorigenesis. lation and inactivation of forkhead transcription factors, Pim kinases have also been implicated to induce cell cycle FoxO1a and FoxO3a, was involved in the transcriptional progression and cell growth. Overexpression of Pim kinases is Kip1 repression of the p27 gene. In contrast, inhibition of Pim reported to promote cell cycle and cell growth, whereas pim signaling by expressing the dominant-negative form of Pim1 knockdown delays them (7, 13, 14). In pim1, pim2, and pim3 triple increased nuclear p27Kip1 level and attenuated cell prolifera- knockout mice, the cell number and body size was decreased (15). tion. Because the CDK inhibitor p27Kip1 plays a crucial role in Pim1 promotes cell cycle progression at the G1-S and G2-M tumor suppression by inhibiting abnormal cell cycle progres- transitions. The G1-S–stimulating phosphatase CDC25A was found sion, Pim kinases promote cell cycle progression and to be a substrate of Pim1, and it has been shown that it could be tumorigenesis by down-regulating p27Kip1 expression at both activated through phosphorylation by Pim1 (16). Furthermore, transcriptional and posttranslational levels. [Cancer Res Pim1 phosphorylated G2-M–stimulating phosphatase CDC25C and 2008;68(13):5076–85] increased its phosphatase activity (17). Moreover, Pim1 was reported to phosphorylate C-TAK1 and inactivate its kinase Introduction activity, resulting in CDC25C activation (18). However, the mechanisms by which Pim kinases stimulate cell cycle progression Human cancer remains a serious disease, and at present, there is and cell growth are not fully understood. Therefore, we tried to still no fully critical chemotherapeutic strategy against it. Tumor- identify novel substrates of Pim1 and to elucidate the mechanism igenesis occurs when proto-oncogenes are activated and tumor by which Pim1 induced cell cycle progression and cell growth. suppressor genes are, frequently, mutated and inactivated. Most of We identified the CDK inhibitor p27Kip1 as a novel substrate of the genes are associated with cell cycle progression and cell growth. Pim kinases. p27Kip1 is known to be a key molecule that modulates Understanding how proto-oncogenes and tumor suppressor genes cell cycle progression at the G1-S transition (19–24). The expression contribute to cancer cell proliferation and tumorigenesis could be of p27Kip1 is regulated at both transcriptional and posttranslational promising for anticancer drug development. levels (25–28). From the observation of p27Kip1 knockout mice, The serine (Ser)/threonine (Thr) kinase pim gene is a proto- p27Kip1 behaved like a tumor suppressor (29). Furthermore, reduced oncogene (1). The pim gene has been identified as a common p27Kip1 expression is frequently observed in most human tumors. integration site of the Moloney murine leukemia virus (2). Pim The reduced expression of p27Kip1 was reported to correlate with tumor progression and poor patient survival (30). Here, we showed that Pim kinases phosphorylated and down-regulated p27Kip1 at the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). posttranslational level. We also showed that Pim kinases repressed Kip1 Requests for reprints: Naoya Fujita, Cancer Chemotherapy Center, Japanese p27 expression at the transcriptional level by inactivating Foundation for Cancer Research, Tokyo 135-8550, Japan. Phone: 81-33570-0468; Fax: Forkhead transcription factors. Moreover, we found the inverse 81-3-3570-0484; E-mail: [email protected]. Kip1 I2008 American Association for Cancer Research. correlation between pim1 and p27 mRNA expression in patients doi:10.1158/0008-5472.CAN-08-0634 with cancer. These results indicate that down-regulation of p27Kip1

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Figure 1. Pim kinases promote the G1-S transition by up-regulating CDK2 activity. A, HEK293T cells were transfected with empty pc5FLAG (Mock) or pc5FLAG- encoding wt-Pim1S (Pim1S). After transfection for 36 h, cells were stained with propidium iodide and analyzed using a flow cytometer. B, the polyclonal K562 cells that had been transfected with empty pc5FLAG (Mock) or pc5FLAG encoding wt-Pim1S (wt-Pim1S) were cultured in normal growth medium. CDK2 proteins were immunoprecipitated from polyclonal K562/Mock or K562/wt-Pim1S cells and were subjected to CDK2 kinase assay, as described in Materials and Methods. The reaction samples were electrophoresed and stained with CBB (right, middle). After staining, the levels of incorporated radioactivity were visualized (right, top) and quantified (left) with Typhoon 9410. The amount of immunoprecipitated CDK2 was also shown (right, bottom). Columns, mean of three independent experiments; bars, SD (left). C, the expression level of endogenous (endo) and exogenous (exo) Pim1S, p27Kip1, and h-actin proteins in polyclonal K562/Mock (Mock) or K562/wt-Pim1S (wt-Pim1S) cells was confirmed by immunoblot analysis.

expression is one of the important mechanisms of Pim-mediated (Ser/Thr) Akt substrate (Cell Signaling Technology), human Topo IIh (BD cell cycle progression and tumorigenesis. Transduction Laboratories), and a-tubulin (Serotec). The membranes were incubated with HRP-conjugated secondary antibody. After washing, the membranes were developed with an enhanced chemiluminescence system (GE Materials and Methods Healthcare). Blots were scanned using an Image Reader LAS-3000 mini (Fuji Reagents and cell culture conditions. Imatinib (Glivec or Gleevec, Film) and were quantified using Multi Gauge software. formerly STI571) was kindly provided by Novartis (Basel, Switzerland). Small interfering RNA design and transfection. The pim1 small LY294002 and G418 were purchased from Sigma. MG132 was purchased interfering RNA (siRNA) sequence can be found in the Supplementary from Wako. Human embryonic kidney 293T cells were cultured in DMEM Materials and Methods. Cells were transfected with the siRNAs using the supplemented with 10% FBS. Human fibrosarcoma HT1080, human LipofectAMINE RNAi MAX (Invitrogen). Kip1 prostate carcinoma 22Rv1 and human myelogenous leukemia K562 cells Purification of recombinant GST-Pim1S and p27 proteins. The were cultured in RPMI 1640 supplemented with 10% FBS. To assess cell details of the purification method used can be found in the Supplementary viability, the MTS assay was employed (Promega). The absorbance was Materials and Methods. In vitro measured at 490 nm with a reference at 690 nm, using a microplate- kinase assay. The details of the method used can be found in spectrophotometer (Benchmark Plus; Bio-Rad). Supplementary Materials and Methods. The reactions were electrophoresed Plasmid construction. Information on the plasmids used can be found and stained with CBB. After staining, the levels of incorporated radioactivity in the Supplementary Materials and Methods. were visualized and quantified with a Typhoon 9410 (GE Healthcare). Transient transfection, immunoprecipitation, and Western blot For the CDK2 assay, cells were solubilized with lysis buffer [0.2% NP40, analysis. HEK293T cells were transfected with the appropriate plasmids 150 mmol/L sodium chloride, 50 mmol/L Tris-HCl (pH 7.6), 1 mmol/L using LipofectAMINE 2000 (Invitrogen). HT1080 cells were transfected phenylmethylsulfonylfluoride, and 1 mmol/L aprotinin] and CDK2 was using FuGENE6 (Roche). K562 cells were transfected using Cell Line immunoprecipitated using an anti-CDK2 antibody (Upstate Biotechnology). Nucleofector Kit V and Amaxa-Nucleofector (Amaxa). The kinase activity in the immunoprecipitated CDK2 was estimated using The preparation of cell lysates for immunoprecipitation and Western blot cdk1/cdc2 kinase assay kit (Upstate Biotechnology). The reactions were analysis and of the whole-cell lysates were done as previously described electrophoresed and stained with CBB. The levels of incorporated (19, 20). The nuclear and cytoplasmic fractions were separated using an radioactivity were visualized and quantified using a Typhoon 9410. NE-PER kit (Pierce). For immunoprecipitation, we used antibodies to p27Kip1 Immunostaining. HT1080 cells were transfected with the appropriate (C-19; sc-528; Santa Cruz Biotechnology), FoxO3a (Cell Signaling Technology), plasmids. After transfection for 24 h, cells were fixed in 4% formaldehyde in or FLAG-tag (clone M2; Sigma). In some experiments, cell lysates were PBS. After washings and permeabilization, the HA-Pim1S was detected by incubated with normal mouse IgG-conjugated agarose (Santa Cruz staining with rat anti-HA antibody, following incubation with Oregon green– Biotechnology) or protein A agarose that had been conjugated with normal conjugated anti-rat antibody (Molecular Probes). The FLAG-p27Kip1 were rabbit IgG. Then, the immunoprecipitated proteins or the cell lysates were detected by staining with rabbit anti-FLAG antibody (Sigma), following electrophoresed and blotted onto a nitrocellulose membrane. The mem- treatment with Alexa Flour 568–conjugated anti-rabbit antibody (Molecular branes were incubated with antibodies to Pim1 (12H8; sc-13513), p27Kip1 Probes). Nuclei were also detected by staining with Hoechst 33342. The (C-19; sc-528), p-p27Kip1 [(Ser10)-R; sc-12939-R], p-p27Kip1 [(Thr187)-R; details for immunostaining have been previously described (19, 20). sc-16324-R], GFP (B-2; sc-9996), actin (C-2-HRP; sc-8432 HRP), 14-3-3h RNA preparation and real-time PCR. Total RNAs from HEK293T or (H-8; sc-1657), 14-3-3u (C-18; sc-7683; Santa Cruz Biotechnology), FoxO3a, K562 cells were extracted using RNeasy Mini Kit (Qiagen). RNA was reverse- phospho-FoxO1 (Thr24)/FoxO3a (Thr32), phospho-FoxO3a (Ser253), phospho- transcribed by First-Strand Synthesis SuperMix (Invitrogen). Then, the www.aacrjournals.org 5077 Cancer Res 2008; 68: (13). July 1, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research amount of p27Kip1 mRNA was quantified by using PCR LightCycler 480 3552, GenBank accession no. AB003688; ref. 26) and pGL4.74 (Promega) as (Roche) and normalized by the amount of glyceraldehyde-3-phosphate internal controls. After transfection, the cells were lysed. Luciferase activity dehydrogenase mRNA. The sequence of the primers for p27Kip1 and was measured using a PicaGene Luciferase assay system (Toyo-Inc. Co., glyceraldehyde-3-phosphate dehydrogenase can be found in the Supplemen- Ltd.) and LB 960 Microplate Luminometer Centro (Berthold). tary Materials and Methods. Flow-cytometric analysis of transfected cells. HEK293T cells were Luciferase assay. HEK293T cells were transfected with the appropriate transfected with the appropriate plasmids. After transfection for 36 h, the plasmids and a pGL4.10 vector encoding p27Kip1 promoter (bases 2873- cells were stained with propidium iodide. Analyses were performed using a

Figure 2. Pim kinases bind to and directly phosphorylate p27Kip1. A, alignment of the amino acid sequence of the Pim consensus sequence with the primary sequences of human p27Kip1 around T157 and T198 residues. Identical residues are denoted by white letters on black background (left). HEK293T, K562, and 22Rv1 cells were transfected with empty pHM6 alone (À) or pHM6 encoding wt-Pim1S (wt) or KD-Pim1S (KD) together with pFLAG-CMV-2 encoding p27Kip1. After transfection for 24 h, the cells were harvested and lysed. The whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies (right). B, HEK293T cells were transfected with empty pc5FLAG alone (À) or pc5FLAG encoding PIm1S (+) together with pHM6-p27Kip1. After transfection for 24 h, the cells were lysed. The FLAG-tag protein was immunoprecipitated from the cell lysates. The immunoprecipitated proteins (IP) and the cell lysates (Input) were subjected to immunoblot analysis with the indicated antibodies (left). The nuclear fraction of K562 and 22Rv1 cells were incubated with protein A agarose that had been conjugated with normal rabbit IgG (IgG), rabbit anti-p27Kip1 antibody (p27Kip1 ), or rabbit anti-FoxO3a antibody (FoxO3a). The immunoprecipitated proteins were subjected to immunoblot analysis with the indicated antibodies (right). C, HEK293T cells were transfected with empty pHM6 alone (À) or pHM6-Pim1S (+) together with empty pFLAG-CMV-2 (À) or pFLAG-CMV-2 encoding wt-p27kip1 or the indicated p27Kip1 mutants. After transfection for 24 h, the cell lysates were electrophoresed and immunoblotted with the indicated antibodies. D, purified recombinant wt-p27Kip1 (lane 1), S10A-p27Kip1 (lane 2), T157A-p27Kip1 (lane 3), T187A-p27Kip1 (lane 4), T198A-p27Kip1 (lane 5), and GST (lane 6) proteins were incubated with GST-tagged recombinant Pim1S for 30 min at 30jC in the presence of [g-32P]ATP. The reaction samples were electrophoresed and stained with CBB (bottom). After staining, the levels of incorporated radioactivity were visualized with Typhoon 9410 (top).

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Figure 3. Pim-mediated p27Kip1 phosphorylation promotes its binding to 14-3-3 and nuclear export. A, HEK293T cells were transfected with empty pHM6 (À, lanes 1 and 2) or pHM6-wt-Pim1S (+, lanes 3–8) together with empty pFLAG-CMV-2 (À, lane 1) or pFLAG-CMV-2 encoding wt-p27kip1 (wt, lanes 2 and 3)or the indicated p27Kip1 mutants. After transfection for 24 h, the cells were harvested, lysed, and immunoprecipitated with an anti-FLAG agarose, as described in Materials and Methods. The immunoprecipitants (IP) and the cell lysates (Input) were subjected to immunoblot analysis with the indicated antibodies. B, HT1080 cells were transfected with pHM6-wt-Pim1S together with pFLAG-CMV-2 encoding wt-p27Kip1 (wt), T157A-p27Kip1 (T157A), T198A-p27Kip1 (T198A), or T157A/ T198A-p27Kip1 (T157A/T198A). After transfection for 24 h, HA-tagged Pim1S proteins were detected by staining with an anti-HA antibody following Oregon green–conjugated anti-rat antibody incubation. The FLAG-tagged p27Kip1 proteins were detected by staining with an anti-FLAG antibody, following treatment with Alexa Flour 568–conjugated anti-rabbit antibody. Nuclei were detected by staining with Hoechst 33342. The cells were washed and then visualized using a fluorescence microscope equipped with a CCD camera (top). The percentage of the cells that exhibited cytoplasmic p27Kip1 was determined by counting 200 transfectants that expressed both Pim1 and p27Kip1 protein in each cell (bottom). C, HEK293T cells were transfected with empty pc5FLAG alone (À)or pc5FLAG encoding PIm1S (+) together with pFLAG-CMV-2 encoding p27Kip1 (+). After transfection for 24 h, the cells were harvested. The cytoplasmic and nuclear fractions were separated, electrophoresed, and immunoblotted with the indicated antibodies. D, HEK293T cells were cotransfected with empty pHM6 alone (À) or pHM6 encoding wt-, T157A- or T198A-p27Kip1 together with pc5FLAG encoding none (À) or wt-Pim1S (+). After transfection for 36 h, the cells were stained with propidium iodide and analyzed with a flow cytometer. www.aacrjournals.org 5079 Cancer Res 2008; 68: (13). July 1, 2008

Cytomics 500 flow cytometer (Beckman Coulter). The details of the method substrate antibody. As shown in Fig. 2A (right), the Akt substrate used have been previously described (31). antibody recognized the phosphorylated form of p27Kip1 only when Expression analysis on cDNA filter array. Cancer Profiling Array II was HEK293T, K562, and 22Rv1 cells were cotransfected with a wt- purchased from Clontech. The details of the method can be found in Pim1S–expressing plasmid but not with a kinase-inactive Pim1S Supplementary Materials and Methods. (KD-Pim1S), indicating that Pim1S kinase activity is required for p27Kip1 phosphorylation. As shown in Supplementary Fig. S1, Results recombinant GST-tagged Pim1S was incubated in vitro with Relationship between Pim1 expression and cell cycle recombinant p27Kip1 and the band of phosphorylated p27Kip1 could progression. A previous study reported that the amount of Pim1S be detected. The phosphorylation was inhibited by adding the increased at the G1-S transition in chronic myelogenous leukemia Pim1 inhibitor LY294002 (3). Pim1S is known to be phosphorylated (CML) BV173 cells (32). We confirmed the increase of endogenous by itself at Ser8 (33). Consistent with this report, Pim1S was Pim1S at the G1-S transition in another CML K562 cells after cell phosphorylated by itself, and the phosphorylation was also synchronization (data not shown). When HEK293T cells were inhibited by LY294002 (Supplementary Fig. S1). These results transfected with a wt-Pim1S–expressing plasmid, decrease in the suggest that Pim1S directly phosphorylates p27Kip1. cell number at the G1 phase and increase in the cell number at Pim kinases were composed of Pim1S/1L, Pim2S/2M/2L, and S phase were observed (Fig. 1A). These results suggested that Pim Pim3 in human (3, 14, 37). As shown in Supplementary Fig. S2, Kip1 could promote the G1-S transition. all variants were able to phosphorylate p27 . In addition, all Kip1 To clarify the mechanisms of G1-S transition, we estimated CDK2 recombinant Pim variants could phosphorylate p27 in vitro kinase activity in the polyclonal K562/wt-Pim1S and K562/Mock (data not shown). These results strongly suggest that p27Kip1 is a cells because CDK2 activity is the key regulator for G1-S transition. substrate of Pim kinases in vivo. We found that CDK2 activity was up-regulated in the Pim1S Identification of T157 and T198 as Pim phosphorylation transfectant but that the amount of CDK2 was unaffected (Fig. 1B). sites in p27Kip1. p27Kip1 is known to be phosphorylated at serine- It has been well-known that CDK2 activity is regulated by other 10 (S10) and threonine-187 (T187) residues, in addition to T157 and regulatory factors. Therefore, we examined whether Pim1S down- T198 (19). To identify the phosphorylation sites, we first tested Kip1 Kip1 regulated the CDK inhibitor p27 expression or not. As shown in whether Pim1S could phosphorylate NH2-terminal–deleted p27 Fig. 1C, Pim1S overexpression down-regulated the expression level mutants DN27 (amino acids 27–198) and DN52 (amino acids 52– Kip1 of p27 . These results suggest that Pim1S stimulates cell cycle 198) and COOH-terminal–deleted p27Kip1 mutants 136STOP Kip1 progression at the G1-S phase, possibly by down-regulating p27 (amino acids 1–136), 156STOP (amino acids 1–156), and 185STOP and activating CDK2. (amino acids 1–185). Although Pim1S phosphorylated wild-type, Kip1 Pim binds to and phosphorylates p27 in vitro and in cells. DN27, DN52, and weakly, 185STOP, Pim1S could not phosphorylate Kip1 The concentration of p27 is known to be transcriptionally and 136STOP or 156STOP, lacking both T157 and T198 residues posttranslationally regulated (25–28). To clarify the mechanisms of (Supplementary Fig. S3). We then prepared four point mutants Kip1 Pim-mediated down-regulation of p27 , we sought to check (S10A, T157A, T187A, and T198A-p27Kip1) in which predicted Kip1 whether p27 was directly phosphorylated by Pim kinases because phosphorylation sites were converted to alanine. As shown in Kip1 overexpression of Pim1S led to a decreased p27 level (Fig. 1C). Fig. 2C, the anti-Akt substrate antibody slightly recognized T157A- Putative substrate target sequences of Pim have been identified p27Kip1 or T198A-p27Kip1 even when cells were cotransfected with using a chemically synthesized peptide library (33, 34). Using an Pim1S. Point mutations at S10 or T187 did not affect the band in silico search, we identified two high homologues to Pim consensus intensities recognized by the anti-Akt substrate antibody. Immu- sequences around threonine-157 (T157) and threonine-198 (T198) noblot analysis using anti–phospho-S10 or anti–phospho-T187 Kip1 residues in p27 (Fig. 2A, left). Then, we investigated the p27Kip1 revealed that Pim1 was not associated with p27Kip1 Kip1 interaction between Pim and p27 by immunoprecipitating phosphorylation at S10 or T187 residues (Fig. 2C). These results Kip1 analysis. Figure 2B (left) shows that HA-tagged p27 was suggest that both T157 and T198 residues are the Pim coimmunoprecipitated with FLAG-tagged Pim1S, suggesting that phosphorylation sites. Consistent with these results, mutation at Kip1 Pim kinases interact with p27 in cells. To confirm that Pim1S T157 or T198 residues decreased Pim1S-mediated p27Kip1 phos- Kip1 forms a complex with p27 at endogenous expression levels, we phorylation in vitro (Fig. 2D). These results strongly indicate that performed the immunoprecipitation following Western blot analysis Pim kinases phosphorylate p27Kip1 at both T157 and T198 residues. using K562 and 22Rv1 cells (Fig. 2B, right) because it has already been Pim kinases stimulate nuclear export of p27Kip1. It has been shown that Pim1S was up-regulated and potentially had a strong role reported that Akt-mediated p27Kip1 phosphorylation at T157 and in the progression of these cancers (7, 8, 35, 36). Endogenous Pim1S T198 residues induced the binding to 14-3-3 protein (19, 20, 22, 38) Kip1 could be detected in endogenous p27 immunoprecipitants and promoted nuclear export of p27Kip1 (19–24, 38). Because Pim (Fig. 2B, right, lanes 2 and 5). These results indicate that Pim1S kinases also promoted p27Kip1 phosphorylation at T157 and T198 Kip1 physiologically interacts with p27 in cells. residues (Fig. 2), we analyzed the interaction between p27Kip1 and We then examined whether Pim kinases directly phosphorylated 14-3-3 after Pim1-mediated phosphorylation. When HEK293T cells Kip1 p27 . The consensus sequence of Pim kinase is very similar to were transfected with FLAG-tagged wt-p27Kip1, p27Kip1 binding to that of Ser/Thr kinase Akt. The commercially available anti– endogenoustotal14-3-3(Fig.3A,top)orendogenous14-3-3u (Fig.(Fig. phospho-Ser/Thr Akt substrate antibody could preferentially (Fig. 3A, second panel) was promoted only by HA-tagged wt-Pim1S recognize the conserved Akt phosphorylation motif (19, 20, 22). expression. As the 14-3-3u mutant (RA), which lost its ligand- In addition, the predicted Pim phosphorylation sites around T157 binding ability (19), failed to bind to p27Kip1 even in the presence of Kip1 and T198 residues in p27 have been reported to be wt-Pim1S (Supplementary Fig. S4), the binding to 14-3-3u became phosphorylated by Akt (19, 20, 22). We thus estimated the Pim- specific. Consistent with the previous reports (19, 20, 22), the mediated phosphorylation at T157 and T198 residues using the Akt interaction between 14-3-3 and p27Kip1 was weakened when the

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Figure 4. Proteasome-dependent degradation and transcriptional repression are both associated with Pim-mediated decrease in p27Kip1 expression. A, HEK293T cells were transfected with empty pc5FLAG alone (À) or pc5FLAG encoding wt-Pim1S (+). After transfection for 24 h, medium was replaced with normal growth medium containing 10 Amol/L of MG132, and the cells were incubated for an additional 3 h. The whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies. B, HEK293T cells were transfected with empty pc5FLAG alone (Mock) or pc5FLAG vector encoding wt-Pim1S (Pim1S). After transfection for 24 h, RNA was extracted from each sample. The amounts of p27Kip1 mRNA were quantified by real-time PCR, as described in Materials and Methods. C, K562 cells were transfected with empty pc5FLAG alone (À) or pc5FLAG encoding wt-Pim1S (+). After transfection for 6 h, 3 Amol/L of imatinib was added to some samples (+). After incubation for an additional 24 h, RNA was extracted from each sample. The amounts of p27Kip1 mRNA were quantified by real-time PCR, as described in Materials and Methods. Columns, mean of experiments done in triplicate; bars, SD.

T157 or T198 residue in p27Kip1 was converted to Ala (T157A or first examined the change in p27Kip1 half-life. Overexpressed Pim1S T198A, respectively). Moreover, mutation at both residues (T157A/ stimulates the decrease in p27Kip1 levels (Supplementary Fig. S5). T198A) could no longer bind to 14-3-3, even when the cells were We next investigated the role of proteasome in Pim1S-mediated cotransfected with wt-Pim1S (Fig. 3A, lane 8). We also checked the down-regulation of endogenous p27Kip1. The decrease in p27Kip1 p27Kip1 binding to other 14-3-3 isoforms (h, q, j, ~, and g) and expression was attenuated by treatment with proteasome inhibitor found that the exogenously expressed 14-3-3 isoforms bound to MG132 (Fig. 4A). However, MG132 could not fully rescue the Pim- p27Kip1-like endogenous 14-3-3 protein did (Supplementary Fig. S4). mediated down-regulation of p27Kip1. These results suggest that These results suggest that Pim kinases promote 14-3-3 binding to Pim kinases down-regulate p27Kip1 level by promoting proteasome- p27Kip1 by phosphorylating at both T157 and T198 residues. dependent degradation and by other mechanisms. Next, we investigated the subcellular localization of p27Kip1. The concentration of p27Kip1 is transcriptionally and posttransla- When HT1080 cells were cotransfected with wt-Pim1S and wt- tionally regulated (25–28). Therefore, we quantitatively evaluated p27Kip1, we could observe the nuclear export of wt-p27Kip1 (Fig. 3B, the change in p27Kip1 mRNA level by real-time PCR. As shown in top) but not that of T157A, T198A, and T157A/T198A-p27Kip1.By Fig. 4B, overexpression of Pim1S in HEK293T cells down-regulated counting, we found that the ratio of cells with cytoplasmic wt- the amount of p27Kip1 mRNA to 50% of controls. Consistent with the p27Kip1 was f80% (Fig. 3B, bottom). However, the percentage of previous report (39), imatinib treatment down-regulated Pim1S level cells that contained cytoplasmic T157A, T198A, and T157A/T198A- and induced p27Kip1 expression in K562 cells in a dose-dependent p27Kip1 was <20%. Immunoblot analysis confirmed that the fashion (Supplementary Fig. S6). Under these conditions, imatinib decrease in nuclear p27Kip1 and the increased cytoplasmic p27Kip1 increased the level of p27Kip1 mRNA (Fig. 4C). The imatinib- were also observed in Pim1S transfectants (Fig. 3C), indicating that mediated increase in p27Kip1 mRNA expression was displaced by Pim-mediated p27Kip1 phosphorylation promoted 14-3-3 binding ectopic expression of Pim1S (Fig. 4C). These results suggest that and nuclear export. To confirm the direct link between p27Kip1 Pim1S down-regulates p27Kip1 level by inducing proteasome- phosphorylation and cell cycle progression, we transfected dependent degradation and by repressing its transcription. Kip1 wt-Pim1S together with wt-p27Kip1 or phosphorylation-resistant Pim kinases repress p27 transcription by phosphorylat- p27Kip1 into HEK293T cells because their transfection efficiency ing and inactivating FoxO transcription factors. It is well was >90% (data not shown). Ectopic expression of wild-type or known that p27Kip1 transcription is regulated by FoxO transcription mutant p27Kip1 in the absence of wt-Pim1S induced cell cycle arrest factors such as FoxO1a, FoxO3a, and FoxO4 (40, 41). In this family, at the G1 phase (Fig. 3D; data not shown). Cotransfection of FoxO3a is reported to contribute mainly to the transcription of the wt-Pim1S rescued cells from wt-p27Kip1–mediated, but not T157A- p27Kip1 gene (27, 40). Using an in silico search, we found that three Kip1 or T198A-p27 –mediated cell cycle arrest at the G1 phase. The residues, threonine-32 (T32), serine-253 (S253), and serine-315 results clearly indicate that Pim kinases promote cell cycle (S315) partially matched the Pim consensus sequence in human progression by phosphorylating and inactivating p27Kip1 in cells. FoxO3a (Fig. 5A, top). Phosphorylation of the residues was reported Promotion of p27Kip1 degradation by Pim kinases. The to reduce FoxO3a’s transcriptional activity by inducing its nuclear cytosolic p27Kip1 was ubiquitinated by ubiquitin ligase KPC1/2 export (40–42). Because we had discovered that endogenous Pim1S following degradation by proteasome at the G1-S phase (25). We interacted with endogenous FoxO3a in cells (Fig. 2B, right, lanes 3 www.aacrjournals.org 5081 Cancer Res 2008; 68: (13). July 1, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research and 6), we examined whether Pim could phosphorylate FoxO3a in mock or H212R-FoxO3a, increased p27Kip1 promoter activity cells. When HEK293T cells were transfected with Pim1S and (Fig. 5C). The increase in luciferase activity by transfecting wt- wt-FoxO3a, we observed the clear bands of phospho-FoxO3a at T32 FoxO3a was suppressed by wt-Pim1S expression. Ectopic expres- and S253 residues (Fig. 5A, bottom). The Pim-mediated phosphor- sion of wt-Pim1S could not attenuate the AAA-FoxO3a–mediated ylation was not observed in AAA-FoxO3a (T32A/S253A/S315A), increase in p27Kip1 reporter activity (Fig. 5C). These results indicate even when Pim1S was overexpressed. Pim1S might regulate FoxO that Pim phosphorylate and inactivate FoxO transcription factors levels by phosphorylation because the expression level of resulting in the suppression of p27Kip1 transcription. wt-FoxO3a, but not AAA-FoxO3a, was diminished by Pim1S To confirm the negative correlation between pim1S and p27Kip1 expression. Moreover, a decrease in endogenous Foxo3a expression mRNA expression in a pathologic condition, we examined their and an increase in endogenous FoxO3a phosphorylation at T32 and expression in some tumor tissue (T) compared with corresponding S253 residues was observed in HEK293T cells (Fig. 5B). We also normal tissue (N) from patients with prostate cancer. It has already confirmed that the Pim1S-mediated phosphorylation of another been suggested that Pim1 has a potentially strong role in these FoxO family member, FoxO1a at T24, S256, and S319, resulted in cancer progressions (8). In tumor tissue from patients with p27Kip1 transcription inhibition (data not shown). These results prostate cancer, pim1S expression levels were relatively higher indicate that Pim1S phosphorylates and down-regulates FoxO than in individual normal tissue; however, p27Kip1 expressions were transcription factors in cells. lower (Fig. 5D). These results suggest that Pim kinases are To confirm the involvement of Pim in FoxO-mediated p27Kip1 associated with tumorigenesis by negatively regulating p27Kip1 transcription, we cloned a fragment of the human p27Kip1 promoter expression. containing the putative FoxO-responsive elements (26) into Inhibition of endogenous Pim signaling increased the pGL4.10 to generate the p27Kip1 reporter vector. In the absence amount of p27Kip1 and attenuated cell proliferation. To assess of Pim1S, transfection of wt-FoxO3a and AAA-FoxO3a, but not the role of endogenous Pim signaling in p27Kip1 expression and cell

Figure 5. Pim kinases down-regulate p27Kip1 transcription by phosphorylating and inactivating FoxO3a. A, alignment of the amino acid sequence of the Pim consensus sequence with the primary sequences of human FoxO3a around T32 and S253 residues. Identical residues are denoted by white letters on black background (top). HEK293T cells were transfected with empty pHM6 alone (À)or pHM6 encoding wt-Pim1S (+) together with empty pc5FLAG alone (À) or pc5FLAG encoding wt-FoxO3a (wt) or AAA-FoxO3a (AAA). After transfection for 24 h, the whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies. Asterisks, the background bands (bottom). B, HEK293T cells were transfected with empty pc5FLAG alone (À) or pc5FLAG encoding wt-Pim1S (+). After transfection for 24 h, the whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies. C, HEK293T cells were transfected with empty pHM6 alone (À)or pHM6 encoding wt-Pim1S (+) together with pc5FLAG encoding none (Mock), wt- FoxO3a (wt), AAA-FoxO3a (AAA), or H212R-FoxO3a (H212R). Cells were also transfected with pGL4.10 vector containing p27Kip1 promoter and pGL4.74 as an internal control. After transfection for 24 h, luciferase activities were calculated as described in Materials and Methods. Columns, mean of experiments done in triplicate; bars, SD. D, cancer profiling Array II containing normalized cDNA from normal tissue (N) or tumor tissue (T) of four patients with prostate cancer was probed with 32P-labeled pim1 and p27Kip1 probes, as described in Materials and Methods. Images were acquired and subsequent data analysis was done using Typhoon 9410. The densitometry units for the pim1 and p27Kip1 probed array were normalized to the densitometry units for a control ubiquitin-probed array.

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Figure 6. Induction of nuclear p27Kip1 expression and inhibition of cell growth by expressing a dominant-negative form of Pim1 or knockdown of Pim1 by siRNA. A, the polyclonal K562 cells that had been transfected with an empty pc5FLAG (K562/ Mock), pc5FLAG encoding wt-Pim1S (K562/wt-Pim1S), or dominant-negative form of Pim1 (K562/DN-Pim1) were cultured in normal growth medium. The total cell lysates were electrophoresed and immunoblotted with the indicated antibodies (top). Expression level of p27Kip1 protein in A was quantified (bottom, left). p27Kip1 mRNA levels in K562/Mock, K562/wt-Pim1S, and K562/DN-Pim1 cells were quantified by real-time PCR method (bottom, right). Columns, mean of experiments done in triplicate; bars, SD. B, 22Rv1 cells were transfected with siRNAs targeting pim1. After transfection for 48 h, the whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies. C, K562/Mock, K562/wt-Pim1S, and K562/DN-Pim1 cells were plated at a low density in triplicate wells. MTS assay was time-dependently performed, as described in Materials and Methods. Points, mean of three independent experiments; bars, SD. D, 22Rv1 cells which transfected with each siRNA were plated at a low density in triplicate wells. Cell number counting was time-dependently performed. Points, mean of three independent experiments; bars, SD.

growth, we generated a dominant-negative form of Pim1 (DN-Pim1; (Fig. 6C). In 22Rv1 cells depleted of Pim1 by siRNA, accumulation of ref. 13). We then established polyclonal K562 cells that had been p27Kip1 was also observed and the growth of the cells was diminished transfected with DN-Pim1 (Fig. 6A, top). The levels of p27Kip1 protein (Fig. 6D). These results suggest that Pim kinases play an important (Fig. 6A, bottom, left) and p27Kip1 mRNA (Fig. 6A, bottom, right) role in cell cycle progression and cell proliferation by regulating were both down-regulated in K562/wt-Pim1S cells although they p27Kip1 expression at transcriptional and posttranscriptional levels. increased in K562/DN-Pim1 cells. In addition, we confirmed that DN-Pim1 had no kinase activity (Supplementary Fig. S7, left). We observed the increase in p27Kip1 expression in nucleus (N) and Discussion the decrease in p27Kip1 expression in cytoplasm (C; Supplementary Pim kinases are overexpressed in patients with lymphoma, Fig. S7, right). Moreover, as shown in Fig. 6B, pim1 gene silencing by leukemia, and prostate cancers (4, 6–8, 26). The ability of Pim to two independent pim1 siRNA resulted in the increase of p27Kip1 stimulate cell growth and inhibit apoptosis may contribute to the expression in 22Rv1 cells. These results confirmed that endogenous promotion of tumorigenesis (3, 5, 13, 31). Previous studies have Kip1 Pim signaling negatively regulated p27 expression in cells. reported that Pim1 stimulates cell cycle progression at the G1-S and Waf1/Cip1 Consistent with this increase, inhibition of Pim signaling by G2-M transitions by phosphorylation of p21 and CDC25A dominant-negative Pim1 (DN-Pim1) diminished the growth of (16, 43) and CDC25C (17, 18). We confirmed that Pim1S over- K562 cells, whereas wt-Pim1S expression promoted cell proliferation expression promoted cell cycle progression at both the G1-S and www.aacrjournals.org 5083 Cancer Res 2008; 68: (13). July 1, 2008

G2-M transitions in HEK293T and K562 cells (Fig. 1A; data not T157 and T198 residues by Pim1 critically contribute to shown). Under these conditions, we found an increase in CDK2 p27Kip1nuclear export and cell proliferation. activity and down-regulation of CDK inhibitor p27Kip1 expression Overexpression of Pim1S decreased p27Kip1 expression (Fig. 1C) Kip1 (Fig. 1B and C). Pim might accelerate cell cycle progression at the G1- and attenuated the half-life of p27 (Supplementary Fig. S5). It is S transition in part by down-regulating p27Kip1 expression levels. well known that p27Kip1 is degraded in cytosol by ubiquitin ligase There are three genes encoding Pim kinases, pim1, pim2, and KPC1/2 at the G1-S transition (25). Thus, nuclear-cytoplasmic Kip1 pim3 (3). It is known that the consensus sequences of Pim1, Pim2, shuttling of p27 by Pim at the G1-S transition may promote its and Pim3 are very similar and that they phosphorylate the same degradation by KPC1/2. Pim-mediated p27Kip1 down-regulation was substrate, such as Bad and 4EBP-1 (14, 33, 34, 44). The depletion of mediated not only by proteasome-dependent degradation (Fig. 4A) all pim1, pim2,andpim3 genes is required for the drastic but also by transcriptional repression (Fig. 4B and C). We identified phenotype of mice (15). In addition, up-regulation of pim2 is FoxO transcription factors as new substrates of Pim (Fig. 5A). Pim1S observed in pim1 knockout mice (45). Thus, Pim1, Pim2, and Pim3 expression induced the phosphorylation of FoxO3a (Fig. 5A and B) could compensate for each function by sharing functional and inactivated its transcriptional activity (Fig. 5C). We also similarity. Consistent with these reports, we showed that all the confirmed that Pim1S phosphorylated FoxO1a and inactivated its Pim variants could phosphorylate p27Kip1 at T157 and T198 transcriptional activity as well (data not shown). A previous report residues (Supplementary Fig. S2). Therefore, p27Kip1 might be an showed that phosphorylation at T32, S253, and S315 residues in important substrate of Pim kinases. FoxO3a induced 14-3-3 binding, nuclear export, and proteasome- We previously reported that T198 residue in p27Kip1 is mediated degradation (42). Consistent with that report, we found phosphorylated by Akt and RSK1/2 (19, 20, 22). These kinases that Pim1S overexpression decreased the exogenous and endoge- are regulated through different pathways. Akt is regulated by the nous FoxO3a expression level (Fig. 5A and B). phosphoinositide-3-kinase pathway and RSK1/2 through the Ras/ In a physiologic condition, Pim expression was regulated by JAK/ Raf/MAPK pathway, whereas Pim is regulated by the JAK/STAT STAT signaling (3). In a pathologic condition, acute myelogenous pathway (3). It is still unclear why three kinases phosphorylate the leukemia (AML), FMS-like tyrosine kinase-3 (FLT3) activation by same residues in p27Kip1. To explain how these kinases coordinate in-frame internal tandem duplications (ITD) was reported to Kip1 their contributions to cell cycle progression at the G1-S transition induce the FoxO3a inactivation (49), leading to p27 down- by down-regulating p27Kip1, we checked the changes in the regulation (36). ITD-FLT3 was shown to stimulate Pim1 expression amounts of Akt and phospho-Akt (T308) after cell synchronization by activating STAT5 (36). Therefore, Pim1 might down-regulate in K562 cells. The amounts were not changed throughout the cell p27Kip1 expression by negatively regulating FoxO3a downstream of cycle, although the amount of Pim1 was drastically increased at ITD-FLT3. In CML, BCR-ABL activates STAT3 and STAT5, leading Kip1 the G1-S transition (data not shown). Therefore, Pim1S mainly to Pim1 expression (39). We detected the increase in p27 Kip1 contributes to p27 down-regulation at the G1-S phase in K562 protein and mRNA expression in K562 treated with the BCR-ABL cells. inhibitor imatinib. We also found that Pim1S transfection repressed Phosphorylation of p27Kip1 has been thought to regulate its p27Kip1 mRNA expression induced by imatinib (Fig. 4C). These concentration and subcellular localization (19). CDK2-dependent results suggested that Pim stimulated cell growth in AML and CML phosphorylation at T187 has been well characterized and has by down-regulating p27Kip1. been shown to be associated with proteasome-dependent In patients with cancer, p27Kip1 is decreased, and its decrease degradation (28, 46). The S10 residue was identified as a major is strongly correlated with a poor prognosis (30). We detected phosphorylation site of p27Kip1 and might regulate the nuclear- the inverse correlation between Pim1S and p27Kip1 mRNA in cytoplasmic shuttling. However, S10 phosphorylation was not prostate cancer (Fig. 5D). A negative correlation between Pim1S sufficient for its nuclear export (47). Rodier and colleagues (48) and p27Kip1 expression was also observed in various cell lines also suggested that another signal might be necessary to direct (Supplementary Fig. S8). In these tumors, Pim may contribute to p27Kip1 to the cytosol. Previous reports have shown that p27Kip1 down-regulation and poor prognosis. Moreover, targeting phosphorylation of p27Kip1 at T157 or T198 residues was Pim1 was shown to impair the survival of hematopoietic cells associated with its binding to 14-3-3 proteins, which drives that express inhibitor-resistant forms of FLT3 and BCR-ABL (35). cytoplasmic localization (19, 20, 22, 38). Consistent with these Thus, inhibiting Pim kinases may contribute to the cure of AML reports, 14-3-3 protein binding to p27Kip1 did not require its and CML in patients resistant to the inhibitors of FLT3 and phosphorylation at both T157 and T198 residues at the same BCR-ABL. Because pim knockout mice do not show serious time (Fig. 3A; Supplementary Fig. S4). 14-3-3 proteins preferen- defects, Pim kinases are druggable targets, and the small tially bound to p27Kip1 that was phosphorylated at the T198 molecule targeting Pim kinases could be a new anticancer drug. residue rather than at the T157 residue. Regarding nuclear export of p27Kip1, phosphorylation at both T157 and T198 Disclosure of Potential Conflicts of Interest residues at the same time might be required for its nuclear None of the authors have conflicts of interest. export because single mutation at either T157 or T198 was enough to abrogate the nuclear export (Fig. 3B). The 14-3-3 proteins were reported to form homodimers or heterodimers Acknowledgments between different isotypes. Binding the dimer to both phosphor- Received 2/20/2008; revised 4/19/2008; accepted 4/21/2008. ylated T157 and phosphorylated T198 might be required for Grant support: Ministry of Education, Culture, Sports, Science, and Technology of Japan 17016012 and 18015008 (T. Tsuruo and N. Fujita), in part by the Kobayashi promoting nuclear export. The nuclear export would be essential Institute for Innovative Cancer Chemotherapy (N. Fujita), and the Vehicle Racing Commemorative Foundation (N. Fujita). for the G1-S transition because Pim1S expression could not Kip1 The costs of publication of this article were defrayed in part by the payment of page overcome the G1 cell cycle arrest induced by T157A-p27 or charges. This article must therefore be hereby marked advertisement in accordance Kip1 Kip1 T198A-p27 (Fig. 3D). Therefore, phosphorylation of p27 at with 18 U.S.C. Section 1734 solely to indicate this fact.

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www.aacrjournals.org 5085 Cancer Res 2008; 68: (13). July 1, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27 Kip1 at the Transcriptional and Posttranscriptional Levels

Daisuke Morishita, Ryohei Katayama, Kazuhisa Sekimizu, et al.

Cancer Res 2008;68:5076-5085.

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