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 Cancer Res 2008; 68: (13). July 1, 2008 5076 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. Pim Phosphorylates and Down-regulates p27Kip1 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