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Cancer Research 67, 9666, October 15, 2007. doi: 10.1158/0008-5472.CAN-07-1050
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Casein Kinase 2–Interacting Protein-1, a Novel Akt Pleckstrin Homology Domain-Interacting Protein, Down-regulates PI3K/Akt Signaling and Suppresses Tumor Growth In vivo

Emi Tokuda1,2, Naoya Fujita1, Tomoko Oh-hara1, Shigeo Sato1, Atsuo Kurata1, Ryohei Katayama1, Toshiki Itoh3, Tadaomi Takenawa3, Kohei Miyazono2 and Takashi Tsuruo1

1 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research; 2 Department of Molecular Pathology, Graduate School of Medicine and 3 Department of Biochemistry, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Requests for reprints: Naoya Fujita, Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6, Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3520-0111, ext. 5421; Fax: 81-3-3570-0484; E-mail: naoya.fujita{at}jfcr.or.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The serine/threonine kinase Akt plays a central role in cell survival and proliferation. Its activation is linked to tumorigenesis in several human cancers. Although many Akt substrates have been elucidated, the Akt-binding proteins that regulate Akt function remain unclear. We report herein having identified casein kinase 2–interacting protein-1 (CKIP-1) as an Akt pleckstrin homology (PH) domain-binding protein with Akt inhibitory function. CKIP-1 formed a complex with each Akt isoform (Akt1, Akt2, and Akt3) via its NH2 terminus. Dimerization of CKIP-1 via its leucine zipper (LZ) motif at the COOH terminus was found to be associated with Akt inactivation because deletion of the LZ motif eliminated Akt inhibitory function, although it could still bind to Akt. Expression of the NH2 terminus–deleted CKIP-1 mutant containing the LZ motif, but lacking Akt-binding ability, induced Akt phosphorylation and activation by sequestering the ability of endogenous CKIP-1 to bind to Akt. Stable CKIP-1 expression caused Akt inactivation and cell growth inhibition in vitro. In addition, the growth of stable CKIP-1 transfectants xenografted into nude mice was slower than that of mock transfectants. These results indicate that CKIP-1, a novel Akt PH domain-interacting protein, would be a candidate of tumor suppressor with an Akt inhibitory function. [Cancer Res 2007;67(20):9666–76]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cell life is precisely regulated by the balance between survival and apoptotic signals. Akt/protein kinase B is the key regulator of many of the signals associated with cell growth, survival, and antiapoptosis (1, 2). Phosphatidylinositide 3-kinase (PI3K), activated by the growth factors or cytokines, produces the phosphoinositides phosphatidilinositol-3,4-bisphosphate [PtdIns(3,4)P2] or phosphatidilinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3; ref. 3]. Akt and 3-phosphoinositide–dependent kinase 1 (PDK1) bind to these lipids through their pleckstrin homology (PH) domains and translocate to the plasma membrane. Then, they meet on the plasma membrane, where the Thr308 residue of Akt is phosphorylated by PDK1 (4). To activate Akt, phosphorylation at both Ser473 and Thr308 of Akt is essential. The kinase responsible for Akt phosphorylation at Ser473 residue was referred to as PDK2. It has been reported that Akt itself, DNA-dependent protein kinase, rictor-mammalian target of rapamycin complex, integrin-linked kinase, and others are candidates for PDK2 (5). The activated Akt phosphorylates many substrates, such as Bad, caspase-9, and forkhead transcription factors (6). Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a negative regulator of the PI3K-Akt pathway (7) because of its ability to dephosphorylate the D3 position of phosphoinositides (8).

The interaction between phosphoinositides and Akt is important for Akt activation (9). The NH2-terminal PH domain of Akt is critical for its binding to phosphoinositides. It has been reported that the PH domain was also involved in the binding to several proteins (10). T-cell leukemia/lymphoma 1 (TCL1) has been reported to promote Akt activity by binding to the Akt PH domain (11, 12). The expression of TCL1 was limited in embryos, germ cells, and lymphocytes, whereas Akt was ubiquitously expressed (13). Thus, we hypothesized that some molecules that interact with the Akt PH domain regulate Akt activity in vivo. We did Escherichia coli two-hybrid screening with the Akt1 PH domain as a bait. We succeeded in identifying casein kinase 2–interacting protein-1 (CKIP-1) as an Akt PH domain-binding protein.

CKIP-1 was first reported as an interactive partner of casein kinase 2{alpha} (CK2{alpha}; ref. 14). CKIP-1 expression is ubiquitous in normal tissues and is induced during muscle differentiation (14, 15). CKIP-1 was composed of a PH domain at the NH2 terminus, a leucine zipper (LZ) motif at the COOH terminus, and five proline-rich motifs throughout the protein. It has been reported that CKIP-1 bound to lipid through its PH domain and dimerized through the LZ motif (15, 16). Overexpressed CKIP-1 localizes in the plasma membrane and partly in the nuclear. The reported binding partners of CKIP-1 were CK2{alpha} and ataxia-telangiectasia mutated (ATM) kinase (14, 17). The interactions between CKIP-1 and CK2{alpha} or ATM kinase caused them to localize to the plasma membrane. When cells underwent apoptosis, CKIP-1 was cleaved by caspase-3 (18). The cleaved CKIP-1 fragment then repressed activator protein-1 activity and promoted apoptosis (18).

Here, we report that CKIP-1 directly binds to the PH domain of Akt and decreases Akt kinase activity. The LZ motif of CKIP-1 plays an important role in the suppression of Akt kinase activity. CKIP-1 leads to the decrease in cell growth in vivo and in vitro. These results indicate that CKIP-1 is a novel Akt-interacting protein with inhibitory function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
BacterioMatch two-hybrid screening. Two-hybrid screening was carried out using BacterioMatch Two-Hybrid System version 1 (Stratagene). The human akt1 PH domain cDNA (6–120 amino acids) was generated by PCR amplification using the forward primer 5'-CTATTGTGAAGGAGGGTTGGCT-3' and the reverse primer 5'-GGAAGTCCATCTCCTCCTCCTC-3' with akt1 cDNA as the template and then it was subcloned into a pBT vector (Stratagene). This construct was used as a bait to screen a human fetal brain cDNA library (Stratagene). Reporter strain competent cells (Stratagene) were cotransformed with the bait and the prey plasmids. Approximately 2 x 106 cotransformants were screened. The cotransformants were selected by kanamycin and ß-galactosidase activity. Positive clones were analyzed using BLAST.4

Cell culture conditions. Human embryonic kidney 293T, human osteosarcoma SaOS-2, and human epithelial carcinoma A431 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Human fibrosarcoma HT1080, human lung adenocarcinoma A549, and human ovary adenocarcinoma (OVCAR-3) were cultured in RPMI 1640 supplemented with 10% FBS. Human prostate cancer PC-3 cells were cultured in Ham's F12K medium supplemented with 10% FBS.

Generation of anti-CKIP-1 polyclonal antibody and plasmids. Polyclonal CKIP-1 antibody was obtained by immunizing rabbits with the synthetic peptide SRPWEKTDKGATYTP (241–256 amino acids), corresponding to the middle domain of the human CKIP-1 protein. The antibody was affinity purified from the immunized rabbit serum using a column linked to the peptide.

The human wt-akt1 and E40K-akt1 cDNA in a pFLAG-CMV-2 vector (Sigma) was established in our laboratory. The akt1 PH mutant was subcloned into a pFLAG-CMV-2 vector from pBT-PH-Akt1. The human wt-akt2 and wt-akt3 cDNAs were cloned from the 293T and HeLa cDNA pools, respectively, and subcloned into a pFLAG-CMV-2 vector. The PH-akt2 and PH-akt3 mutants were cloned using the forward primer 5'-GACTGCTCACTCGCGGATGCTG-3' and the reverse primer 5'-GCTGCTTGAGGCTGTTGGCGAC-3' for PH-akt2 and the forward primer 5'-TGAGCGATGTTACCATTGTGAAAGAAGGTTGGG-3' and the reverse primer 5'-GTGCCTTTACCTAGTAGTTTC-3' for PH-akt3 and subcloned into a pFLAG-CMV-2 vector. The human wt-CKIP-1 was generated by PCR amplification using forward primer 5'-TGATGAAGAAGAACAATTCCGCCAAGCGG-3' and the reverse primer 5'-CCCTCACATCAGGCTCTTCCGGTAC-3' from the MGC clone plasmid (Invitrogen) as a template and subcloned into a pHM6 vector (Roche), pQBI 50-fC3 vector (Wako), pGEX-6P-3 vector (GE Healthcare), or pLPCX vector (Clontech). The {Delta}N199-CKIP-1 mutant was cloned using the forward primer 5'-CAACCTCTTGTGCTGAGAGCTTTC-3' and the reverse primer 5'-CCCTCACATCAGGCTCTTCCGGTAC-3' and subcloned into a pHM6 vector. The {Delta}C146-CKIP-1, {Delta}C201-CKIP-1, and {Delta}C345-CKIP-1 mutants were generated from wt-CKIP-1 as a template by converting the appropriate stop codons using the QuikChange Site-Directed Mutagenesis kit (Stratagene). The 200-343-CKIP-1 was generated from {Delta}N199-CKIP-1 as a template by converting the Pro344 codon to a stop codon using the QuikChange Site-Directed Mutagenesis kit. KRW-CKIP-1 was generated from wt-CKIP-1 as a template using the QuikChange Site-Directed Mutagenesis kit. First, the Trp123 codon in wt-CKIP-1 was mutated to the alanine codon using the forward primer 5'-GAAGAGAAGGAATCGGCGATCAATGCCCTC-3' and the reverse primer 5'-GAGGGCATTGATCGCCGATTCCTTCTCTTC-3'. The Lys42 and Arg44 codons in W123A-CKIP-1 were further converted to cysteine codons to establish the KRW-CKIP-1 mutant using the forward primer 5'-GGGAGATTTGGTGCAACTGCTATGTGGTGC-3' and the reverse primer 5'-GCACCACATAGCAGTTGCACCAAATCTCCC-3'.

Transient and stable transfection. Cells were transfected with the appropriate plasmids using SuperFect transfection reagent (Qiagen) or LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturers' instructions. The stable transfectants were selected by cultivating them in medium containing 400 µg/mL geneticin (Sigma).

Immunoprecipitation and immunoblotting. Cells were harvested and solubilized in lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 0.2% NP40, 10% glycerol, 1 mmol/L EDTA, 1.5 mmol/L magnesium chloride, 137 mmol/L sodium chloride, 50 mmol/L sodium fluoride, 1 mmol/L sodium vanadate, 12 mmol/L ß-glycerophosphate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L aprotinin] or SDS buffer [20 mmol/L Tris-HCl (pH 7.5), 1% SDS, 10% glycerol]. Tagged proteins were immunoprecipitated with an anti-FLAG agarose (Sigma) or an anti-hemagglutinin (HA) agarose (Sigma). Endogenous Akt was immunoprecipitated with an anti-Akt agarose (C-20; Santa Cruz Biotechnology) or normal goat IgG agarose. Then, the immunoprecipitated proteins or the cell lysates were electrophoresed and blotted onto a nitrocellulose membrane. The membranes were first incubated with antibodies to FLAG-horseradish peroxidase (HRP; Sigma), HA-HRP (Roche), Auto Fluorescent Protein (Wako), Akt, phosphorylated Akt (Ser473), phosphorylated Akt (Thr308), PDK1, FKHRL1, phosphorylated FKHR (Thr24)/FKHRL1 (Thr32; Cell Signaling Technology), glycogen synthase kinase (GSK)-3, phosphorylated GSK (Ser21), glutathione S-transferase (GST)-HRP (Upstate Biotechnology), p27 (C-19), or ß-actin (C-2; Santa Cruz Biotechnology). Subsequently, membranes were washed and incubated with HRP-conjugated secondary antibody. After washing, the membranes were developed with an enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (GE Healthcare).

Immunofluorescence microscopy. PC-3 cells were fixed with 4% paraformaldehyde for 10 min at 23°C and then treated with PBS containing 0.1% Triton X-100, 3% bovine serum albumin, and 10% gammagard (Baxter) for 1 h at 4°C. Cells were incubated with the generated rabbit anti-CKIP-1 antibody (1:50) and mouse anti-Akt1 monoclonal antibody (clone 2H10; 1:100; Cell Signaling Technology) as primary antibodies for 2 h at 4°C. Then, the cells were incubated with an Alexa Fluor 568–conjugated anti-rabbit IgG antibody (1:1,000) and an Alexa Fluor 488–conjugated anti-mouse IgG antibody (1:1,000) as secondary antibodies for 1 h at 4°C. After washing, the cells were visualized using a fluorescence microscope (Olympus FV1000 confocal microscope) equipped with a charge-coupled device camera.

In vitro Akt kinase assay. Akt kinase assay was carried out using an Akt IP-Kinase Assay kit (Upstate Biotechnology). Immunopurified FLAG-tagged Akt was incubated with 100 µmol/L Akt substrate peptide, 10 µmol/L protein kinase A inhibitor peptide, magnesium/ATP cocktail (16.8 mmol/L magnesium, 112.5 µmol/L ATP), and 1 µCi/µL [{gamma}-32P]ATP for 30 min at 30°C. Reaction was stopped by adding 40% trichloroacetic acid solution. The supernatants were spotted onto P81 phosphocellulose paper, washed thrice with 0.75% phosphoric acid, air dried, and subjected to Cerenkov counting. The measurements were calculated and graphed to a relative value.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To assess cell proliferation, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used. In brief, HT1080 parent, mock transfectant (mock-1 or mock-8), or CKIP-1 transfectant (CKIP-1-16 or CKIP-1-28) cells were incubated with MTT for 3 h at 37°C. Formazan products were solubilized with DMSO, and the absorbance was measured at 525 nm using a microplate spectrophotometer (Benchmark Plus, Bio-Rad). The measurements were calculated and graphed to a relative value.

Purification of recombinant GST-tagged protein. Cultures of BL21 Star E. coli (Invitrogen) containing a pGEX-6P-3 plasmid encoding mock or wt-CKIP-1 were induced for 4 h with 1 mmol/L isopropyl-L-thio-B-D-galactopyranoside at 30°C with shaking. The cells were harvested and solubilized in BugBuster lysis buffer (Novagen) and purified using a Glutathione Sepharose 4B column (GE Healthcare). Recombinant proteins were eluted by adding 50 mmol/L glutathione and dialyzed with PBS.

Pull-down assay. Five micrograms of GST or GST-tagged CKIP-1 were incubated with 3 µg of His-tagged Akt1 or Akt2 (Upstate Biotechnology) in lysis buffer for 4 h at 4°C. Proteins were pulled down with glutathione-Sepharose. The precipitated proteins were eluted by adding 2x SDS sample buffer [20% glycerol, 130 mmol/L Tris (pH6.8), 6% SDS, 0.02% bromphenol blue, 5% 2-mercaptoethanol] and detected by immunoblotting with antibodies to GST or Akt.

Cosedimentation assay. Synthetic liposomes contain 48% phosphatidylcholine (Cell Signals), 48% phosphoethanolamine (Cell Signals), and 4% of the test lipids (mol%; Cell Signals). Liposomes were resuspended at a concentration of 1 mmol/L test lipid in 100 mmol/L HEPES (pH 7.5), 100 mmol/L NaCl, 5 mmol/L EGTA, and 50 mmol/L sucrose and frozen (liquid nitrogen) and thawed (water bath at 37°C) and then sonicated. Ten micrograms of the protein and liposomes were mixed in 10 mmol/L HEPES at pH 7.5, 100 mmol/L KCl, 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.2 mmol/L ATP, and 0.1% Tween 20 and incubated at 23°C for 15 min. After centrifuging at 20,000 x g for 30 min, the vesicle pellets and supernatants were suspended in SDS sample buffer, and bound protein was identified by immunoblotting. The blots were quantified using MultiGauge version 3.0 software and graphed to a relative value.

In vivo tumorigenicity. Parent or stable CKIP-1–transfected HT1080 cells were concentrated to 1 x 107/100 µL. The cells were injected into 6-week-old female BALB/c-nu/nu (nude) mice (Charles River Laboratories). Tumor size was measured twice weekly with a caliper, and tumor volumes were defined as (longest diameter) x (shortest diameter)2 / 2. To confirm the expression of transfected CKIP-1 in tumor samples, the isolated tumors were stabilized in lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 0.2% NP40, 10% glycerol, 1 mmol/L EDTA, 1.5 mmol/L magnesium chloride, 137 mmol/L sodium chloride, 50 mmol/L sodium fluoride, 1 mmol/L sodium vanadate, 12 mmol/L ß-glycerophosphate, 1 mmol/L PMSF, 1 mmol/L aprotinin]. The tumor lysates were electrophoresed and immunoblotted. All animal procedures were done in the animal experiment room of the Japanese Foundation for Cancer Research (JFCR) using protocols approved by the JFCR Animal Care and Use Committee.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of CKIP-1 as an Akt PH domain-binding protein. To identify proteins bound to the PH domain of Akt, we did E. coli–based two-hybrid screening using the PH domain of human Akt1 as the bait. We screened a human fetal brain cDNA library. From the 2 x 106 cotransformants screened, seven candidates were identified. One of the seven encoded full-length cDNA of CKIP-1.

To investigate direct binding between CKIP-1 and Akt, we first carried out a pull-down assay using the recombinant proteins. CKIP-1 was purified from bacteria as a GST fusion protein. The GST-tagged CKIP-1 or GST alone was incubated with recombinant Akt1 or Akt2. Pull-down analysis revealed that both Akt1 and Akt2 formed a complex with GST-CKIP-1 in vitro but not with GST alone (Fig. 1A ). We next examined their interaction in mammalian cells. The HA-tagged CKIP-1 was transfected into 293T cells together with a pFLAG-CMV-2 vector encoding none (mock), Akt1, Akt2, or Akt3. After transfection for 24 h, Akt proteins were immunoprecipitated with an anti-FLAG antibody. As shown in Fig. 1B (left), CKIP-1 could be detected in all Akt isoform immunoprecipitants. By generating Akt PH domain mutants, we found that CKIP-1 also formed a complex with the PH domains derived from all Akt isoforms (Fig. 1B, right). To estimate the endogenous binding, we raised a polyclonal antibody by immunizing rabbits with the peptide comparable with amino acids 241 to 256 of CKIP-1. The generated CKIP-1 antibody recognized both endogenous and exogenous CKIP-1 proteins in HT1080 and 293T cells (Supplementary Fig. S1A, None). When the anti-CKIP-1 antibody was preincubated with the immunizing peptide, the antibody could no longer detect endogenous and exogenous CKIP-1 proteins (Supplementary Fig. S1A, Immunizing peptide). When the mixture of HA-tagged CKIP-1–transfected HT1080 cells and empty vector–transfected HT1080 cells was stained with anti-HA and anti-CKIP-1 antibodies, the staining pattern of anti-CKIP-1 antibody was the same as that of anti-HA antibody (Supplementary Fig. S1B). The result suggests that the generated antibody could specifically recognize CKIP-1. Using the antibody, we could detect endogenous CKIP-1 protein in endogenous Akt immunoprecipitants (Fig. 1C). Then, we checked endogenous CKIP-1 and Akt localization in PC-3 cells. CKIP-1 and Akt were colocalized in the nucleus and the plasma membrane (Fig. 1D). These results indicate that CKIP-1 is an Akt PH domain-binding protein.


Figure 1
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  		Figure 1. CKIP-1 interacts with PH domain of Akt. A, GST pull-down assay. GST alone or GST-tagged CKIP-1 was incubated with His-tagged recombinant Akt1 (lanes 1 and 2) or Akt2 (lanes 3 and 4). GST proteins were pulled down with glutathione-Sepharose. The precipitated proteins (top and second panels) and the input proteins (third and bottom panels) were detected by immunoblotting with antibodies to GST or Akt. B, 293T cells were transfected with pFLAG-CMV-2 vector encoding none (mock), Akt1, Akt2, or Akt3 (lanes 1–4, respectively; left) or the PH domains of Akt1 (Akt1 PH), Akt2 (Akt2 PH), and Akt3 (Akt3 PH; lanes 1–4, respectively; right) together with pHM6-wt-CKIP-1. After transfection for 24 h, the FLAG-tagged Akt was immunoprecipitated with an anti-FLAG antibody-conjugated agarose. The immunoprecipitants (top and middle panels) and cell lysates (bottom panel) were electrophoresed and immunoblotted with antibodies to FLAG or HA. C, HT1080 cell lysates were incubated with normal goat IgG-conjugated agarose (control IgG; lane 2) or anti-Akt antibody-conjugated agarose (lane 3). The immunoprecipitants and cell lysates (input; lane 1) were electrophoresed and immunoblotted with antibodies to CKIP-1 or Akt. IP, immunoprecipitation. D, confocal microscopic analysis showed that endogenous CKIP-1 and endogenous Akt proteins in PC-3 cells were observed in red and green, respectively, by staining with an anti-CKIP-1 antibody (CKIP-1) and an anti-Akt antibody (Akt). Merge represents an overlay of CKIP-1 in red and Akt in green.

 
Down-regulation of Akt phosphorylation by CKIP-1 expression. Because Akt bound to CKIP-1 through its PH domain, CKIP-1 might positively or negatively affect Akt phosphorylation and activation by regulating phosphoinositide binding to the PH domain of Akt. We transfected HA-tagged CKIP-1 into 293T cells together with FLAG-tagged Akt. Immunoblot analysis of cell lysates revealed that CKIP-1 expression markedly reduced the phosphorylated Akt levels at Thr308 and Ser473 in a dose-dependent fashion (Fig. 2A ). Consistent with the decrease in phosphorylated Akt levels, CKIP-1 expression down-regulated the phosphorylation of GSK-3{alpha}, an Akt substrate, in a dose-dependent fashion (Fig. 2A). CKIP-1 expression also decreased the phosphorylation of another Akt substrate, FKHRL1/FoxO3a (Fig. 2B). The effect of CKIP-1 on Akt inhibitory function was also observed in cells transfected with active Akt mutant (E40K-Akt; Fig. 2B).


Figure 2
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  		Figure 2. CKIP-1 expression down-regulates phosphorylation of Akt at Thr308 and Ser473 residues. A, 293T cells were transfected with a pHM6 vector encoding none (–; lane 1) or wt-CKIP-1 (0.1, 0.2, 0.5, 1.0, and 1.5 µg; lanes 2–6, respectively) together with pFLAG-CMV-2-Akt1. After transfection for 24 h, the cell lysates were immunoblotted with the indicated antibodies. B, 293T cells were transfected with a pHM6 vector encoding none (–; lanes 1, 3, and 5) or wt-CKIP-1 (lanes 2, 4, and 6) together with pFLAG-CMV-2 vector encoding none (mock; lanes 1 and 2), wt-Akt1 (wt; lanes 3 and 4), or E40K-Akt1 (E40K; lanes 5 and 6). After transfection for 24 h, the cell lysates were immunoblotted with the indicated antibodies.

 
To confirm the effects of CKIP-1 expression on endogenous Akt phosphorylation, we transfected CKIP-1 using a retrovirus infection system into OVCAR-3 cells. After 48 h of infection, CKIP-1 could be clearly detected in CKIP-1 retrovirus-infected cells (Supplementary Fig. S2A, lane 2). Under this condition, decrease in Akt phosphorylation was observed when compared with the cells infected with a retrovirus pLPCX vector encoding none (mock). Moreover, we transfected HA-tagged CKIP-1 into 293T cells. Immunoblot analysis of cell lysates revealed that CKIP-1 expression decreased endogenous phosphorylated Akt levels and increased the amount of p27Kip1 (Supplementary Fig. S2B). Previous studies showed that Akt pathway regulated p27Kip1 expression. Forkhead transcription factors, which are well-known Akt substrates, have been reported to up-regulate p27Kip1 transcription (19). Akt directly phosphorylates p27Kip1, resulting in facilitation of cytoplasmic localization and degradation (20). These results suggest that CKIP-1 negatively regulates Akt phosphorylation and its kinase activity.

Characterization of domains in CKIP-1. To determine the critical regions in CKIP-1 associated with Akt binding, we generated several CKIP-1 deletion mutants (Fig. 3A ). Immunoprecipitation following immunoblot analysis revealed that Akt bound to COOH-terminal deletion mutants ({Delta}C146, {Delta}C201, and {Delta}C344-CKIP-1) but not to NH2-terminal deletion mutant {Delta}N199-CKIP-1 (Fig. 3B). Thus, the NH2 terminus of CKIP-1 is identified as domain critical for its interaction with Akt. Then, we examined the effects of the mutants on the Akt phosphorylation levels. 293T cells were transfected with FLAG-tagged Akt together with HA-tagged mock or CKIP-1 deletion mutants (Fig. 3C). Consistent with the results shown above, expression of wt-CKIP-1 reduced the phosphorylation levels of Akt (Fig. 3C, lane 2). CKIP-1 mutants lacking the COOH terminus ({Delta}C) showed no effect on Akt phosphorylation levels (Fig. 3C, lanes 3–5), although they could form a complex with Akt. These results indicate that the LZ motif in CKIP-1 is essential for CKIP-1–mediated Akt inhibition.


Figure 3
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  		Figure 3. CKIP-1 interacts with Akt via its NH2-terminal domain. A, schematic diagram of CKIP-1 and CKIP-1 deletion mutants used in the experiments. B, 293T cells were transfected with pFLAG-CMV-2-Akt1 together with a pHM6 vector encoding none (mock; lanes 1 and 2), wt-CKIP-1 (lane 3), or the indicated CKIP-1 mutants (lanes 4–7, respectively). After transfection for 24 h, the HA-tagged proteins were immunoprecipitated with an anti-HA antibody-conjugated agarose. The immunoprecipitants (top and middle panels) and cell lysates (bottom panel) were electrophoresed and immunoblotted with the indicated antibodies. C, 293T cells were transfected with pFLAG-CMV-2-Akt1 together with a pHM6 vector encoding none (mock; lanes 1 and 2), wt-CKIP-1 (lane 3), or the indicated CKIP-1 mutants (lanes 4–6, respectively). After transfection for 24 h, the cell lysates were electrophoresed and immunoblotted with the indicated antibodies. D, 293T cells were transfected with pFLAG-CMV-2-Akt1 together with a pHM6 vector encoding none (mock) or the {Delta}N199 CKIP-1 mutant. After transfection for 24 h, the FLAG-tagged Akt was immunoprecipitated and an Akt kinase assay was carried out. The kinase activity of the mock immunoprecipitant was adjusted as 1 unit. Columns, mean of triplicate experiments; bars, SD.

 
Surprisingly, transfection of {Delta}N199-CKIP-1, lacking Akt-binding ability, increased the Akt phosphorylation level (Fig. 3C, lane 6). To confirm the increase in Akt kinase activity, we did an in vitro kinase assay with the GSK-3 peptide as a substrate. As shown in Fig. 3D, overexpression of {Delta}N199-CKIP-1 resulted in a 2-fold increase in Akt kinase activity. Therefore, {Delta}N199-CKIP-1 could function as a dominant-negative mutant of wt-CKIP-1.

Dimerization via the LZ motif is essential for exhibiting Akt inhibitory function of CKIP-1. It has been reported that CKIP-1 dimerizes by itself through its LZ motif (16). Thus, we first investigated whether CKIP-1 deletion mutants could form a complex with wt-CKIP-1. To do the experiment, we generated blue fluorescence protein (BFP)-tagged wt-CKIP-1 by subcloning CKIP-1 cDNA into pQBI 50-fC3 vector. Then, 293T cells were transfected with BFP-tagged wt-CKIP-1 together with HA-tagged CKIP-1 mutants. After transfection for 24 h, CKIP-1 mutant proteins were immunoprecipitated with an anti-HA antibody-conjugated agarose. Consistent with a previous report (16), HA-tagged wt-CKIP-1 formed a complex with BFP-tagged wt-CKIP-1 (Fig. 4A, lane 2 ). The {Delta}C mutants lacking the LZ motif ({Delta}C146, {Delta}C201, and {Delta}C344-CKIP-1) could not bind to BFP-tagged wt-CKIP-1 (Fig. 4A, lanes 3–5). Meanwhile, {Delta}N199-CKIP-1, having the LZ motif, bound to BFP-tagged wt-CKIP-1 (Fig. 4A, lane 6). To prove the role of the LZ motif in homodimerization, we generated {Delta}N199-CKIP-1 that lacked the LZ motif (200-343-CKIP-1) by converting the Pro344 codon in {Delta}N199-CKIP-1 to a stop codon. The 200-343-CKIP-1 could not form a complex with BFP-tagged wt-CKIP-1 (Fig. 4B, lane 4). Thus, we examined the effect of this mutant on Akt phosphorylation. 293T cells were transfected with FLAG-tagged Akt together with HA-tagged CKIP-1 mutants. The cell lysates were immunoblotted, and we quantified the phosphorylated Thr308 Akt/total Akt (Fig. 4C; data not shown). Although {Delta}N199-CKIP-1 expression promoted a 1.5-fold increase in Akt phosphorylation (Fig. 4C, lane 4) when compared with mock transfectants (Fig. 4C, lane 2), LZ motif deletion from {Delta}N199-CKIP-1 resulted in the loss of Akt phosphorylation-promoting activity (Fig. 4C, lane 5). Therefore, {Delta}N199-CKIP-1 promoted Akt phosphorylation without interacting with Akt. These results suggest that {Delta}N199-CKIP-1 might neutralize the Akt inhibitory function of endogenous wt-CKIP-1 by complex formation via its LZ motif.


Figure 4
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  		Figure 4. The LZ domain of CKIP-1 is essential for homodimerization and Akt inhibitory activity of CKIP-1. A and B, 293T cells were transfected with pQBI 50-wt-CKIP-1 (BFP-CKIP-1) together with a pHM6 vector encoding none (mock; lanes 1), wt-CKIP-1 (lanes 2), or the indicated CKIP-1 mutants. The HA-tagged proteins were immunoprecipitated with an anti-HA antibody-conjugated agarose. The immunoprecipitants (top and middle panels) and cell lysates (bottom panel) were electrophoresed and immunoblotted with antibodies to BFP or HA. C, 293T cells were transfected with pFLAG-CMV-2-Akt1 together with a pHM6 vector encoding none (mock; lanes 1 and 2), wt-CKIP-1 (lane 3), or the indicated CKIP-1 mutants (lanes 4 and 5). The HA-tagged proteins were immunoprecipitated with an anti-HA antibody-conjugated agarose. The immunoprecipitants (fourth and bottom panels) and cell lysates (top, second, and third panels) were electrophoresed and immunoblotted with the indicated antibodies. D, 293T cells were transfected with pQBI 50-CKIP-1 (BFP-wt-CKIP-1) together with pFLAG-CMV-2 vector encoding none (–; lane 1) or Akt1 (lanes 2 and 3) and a pHM6 vector encoding none (–; lanes 1 and 2) or {Delta}N199 CKIP-1 (lane 3). The FLAG-tagged proteins were immunoprecipitated with an anti-FLAG antibody-conjugated agarose. The immunoprecipitants (top and second panels) and cell lysates (third to bottom panels) were electrophoresed and immunoblotted with the indicated antibodies.

 
To investigate whether {Delta}N199-CKIP-1 binds to endogenous full-length CKIP-1 and competes its function to suppress Akt activity, 293T cells were transfected with BFP-tagged wt-CKIP-1, FLAG-tagged mock, or Akt together with HA-tagged mock or {Delta}N199-CKIP-1. The BFP-tagged wt-CKIP-1 could be detected in anti-FLAG immunoprecipitants (Fig. 4D, lane 2). The interaction between FLAG-tagged Akt and BFP-tagged wt-CKIP-1 was attenuated by {Delta}N199-CKIP-1 expression (Fig. 4D, lane 3). In contrast, Akt phosphorylation was clearly induced by {Delta}N199-CKIP-1 expression. The results suggest that {Delta}N199-CKIP-1 enhances Akt activity by interrupting endogenous wt-CKIP-1 binding to Akt via its LZ motif.

CKIP-1 does not compete with Akt for phosphoinositide binding. Akt has a PH domain at the NH2 terminus and binds to PtdIns(3,4)P2 (21) and PtdIns(3,4,5)P3 (22, 23) through the domain. The interaction facilitates Akt translocation to the plasma membrane and its activation (9). CKIP-1 also contains a PH domain at the NH2 terminus. The PH domain was reported to be involved in phosphoinositide binding (15, 16). Therefore, it is possible that CKIP-1 suppresses Akt activation by competing with Akt for binding to phosphoinositides. We first examined the phosphoinositide-binding capability of CKIP-1 using a cosedimentation assay with liposomes. Each liposome was incubated with recombinant GST-tagged CKIP-1 and then sedimented by centrifugation at 20,000 x g. Clearly shown in Fig. 5A , most of the GST-tagged CKIP-1 was cosedimented with liposomes containing phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], or PtdIns(3,4,5)P3. Quantification of the band intensities confirmed the decrease in phosphoinositide-unbound CKIP-1 in the supernatant (Fig. 5A, bottom). Therefore, among the phosphoinositides examined, CKIP-1 preferentially bound to PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3.


Figure 5
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  		Figure 5. CKIP-1 binding to phosphoinositides is not required for CKIP-1–mediated Akt inhibition. A, immunoblotting: liposomes containing the indicated phosphoinositides [phosphatidylcholine (PC)/phosphoethanolamine (PE)/phosphoinositides = 1/1/0.2] were mixed with GST-tagged CKIP-1 (10 µg) and then cosedimented. Precipitates (P) or supernatants (S) were electrophoresed and immunoblotted with an anti-GST antibody. Graph: quantitative analysis of immunoblots. The unbound GST-CKIP-1 protein levels were quantified by MultiGauge version 3.0 software. Relative unbound protein (S) levels were determined by normalizing ECL signals of supernatant intensities to those of supernatant + precipitate intensities. The amount of GST-CKIP-1 at the control (phosphatidylcholine/phosphoethanolamine) was normalized to 100% and graphed as a relative value. B, 293T cells were transfected with pFLAG-CMV-2-Akt1 together with a pHM6 vector encoding none (mock; lanes 1 and 2), wt-CKIP-1 (lane 3), or KRW-CKIP-1 (lane 4). After transfection for 24 h, the cell lysates were electrophoresed and immunoblotted with the indicated antibodies. C, 293T cells were transfected with pFLAG-CMV-2-Akt together with a pHM6 vector encoding none (mock), wt-CKIP-1, or KRW-CKIP-1 mutant. After transfection for 24 h, the FLAG-tagged Akt was immunoprecipitated and an Akt kinase assay was carried out. The Akt kinase activity of the mock immunoprecipitant was normalized as 1 unit. Relative decrease in Akt kinase activity. Columns, mean of triplicate experiments; bars, SD.

 
It has been reported that the mutation in the PH domain of CKIP-1 (K42C/R44C/W123A-CKIP-1; KRW-CKIP-1) resulted in phosphoinositide-binding capability loss and the inability to translocate to the plasma membrane (16). Thus, we generated KRW-CKIP-1 and examined its Akt inhibitory function. 293T cells were transfected with FLAG-tagged Akt (Fig. 5B, lanes 2–4) together with HA-tagged mock (lanes 1 and 2), wt-CKIP-1 (lane 3), or KRW-CKIP-1 (lane 4). Expression of KRW-CKIP-1 decreased the Akt phosphorylation levels, as wt-CKIP-1 did. Moreover, the in vitro kinase assay confirmed the decrease in Akt kinase activity by KRW-CKIP-1 transfection (Fig. 5C). These results indicate that the ability of CKIP-1 to interact with phosphoinositides is not essential for its Akt inhibitory function.

Decrease in in vivo cell growth by CKIP-1 expression. We next examined expression levels of CKIP-1 mRNA in normal and tumor tissues using Cancer Profiling Array II. Decrease in CKIP-1 expression was observed in some tumor tissues when compared with the corresponding normal tissues (Supplementary Fig. S3). We also examined the relationship between endogenous CKIP-1 expression and Akt phosphorylation levels in various cell lines. As shown in Supplementary Fig. S4, we found an inverse correlation between CKIP-1 expression and Akt phosphorylation. Then, we addressed whether introducing CKIP-1 into tumor cells affected Akt kinase activity and cell growth. We used human fibrosarcoma cell line HT1080 because it is highly tumorigenic in vivo. HT1080 cells were stably transfected with the pHM6 vector containing none (mock) or wt-CKIP-1 cDNA. We established several stable transfectants and examined their endogenous Akt phosphorylation levels. The Akt phosphorylation levels were decreased in the stable CKIP-1 transfectants compared with that in parent or mock transfectants (Fig. 6A ). Consistent with the transient transfection analysis in 293T cells (Supplementary Fig. S2B), p27Kip1 expression was increased in the stable CKIP-1 transfectants (Fig. 6A). Because Akt facilitates cell growth by phosphorylating and inactivating cyclin-dependent kinase (CDK) inhibitors, such as p21Cip1 or p27Kip1 (19, 20, 24), we examined the change in growth rate of these transfectants using a MTT assay. The growth rate of the stable CKIP-1 transfectants was remarkably reduced (40–65%) compared with that of the parent or mock transfectants (Fig. 6B). Next, we confirmed that growth delay of stable CKIP-1 transfectants was as a consequence of Akt suppression. Stable CKIP-1 transfectants were further transfected with the pLPCX-E40K-Akt. The cells were selected by adding 0.5 µg/mL puromycin for 2 weeks and then estimated the growth rate. E40K-Akt expression rescued the growth retardation by CKIP-1 expression (Supplementary Fig. S5). These results suggest that CKIP-1 expression down-regulates Akt activity, resulting in cell growth inhibition.


Figure 6
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  		Figure 6. CKIP-1 expression suppresses tumor growth in vivo. A, the cell lysates from parental HT1080 cells (parent) or HT1080 cells that had been stably transfected with a pHM6 vector encoding none (mock-1 and mock-8) or pHM6-CKIP-1 (wt-16, wt-23, wt-28, and wt-42) were electrophoresed and immunoblotted with the indicated antibodies. B, the parent ({square}), mock-1 ({Delta}), mock-8 ({circ}), CKIP-1-16 (bullet), or CKIP-1-28 ({blacktriangleup}) clones were seeded onto a 24-well plate at 5 x 103 cells per well. After the appropriate time points, a MTT assay was done. Points, mean of triplicate experiments; bars, SD. C, the parent or stable transfectants were injected into BALB/C-nu/nu (nude) mice. Eight days after injection, mice were photographed. D, left, tumor lysates from two different mice that had been injected with parent (lanes 1 and 2) or stable transfectants (lanes 3–8) were electrophoresed and immunoblotted with the indicated antibodies; right, tumor volumes from nude mice that had been s.c. injected with parent (bullet), mock-8 ({blacktriangleup}), CKIP-1-16 ({circ}), or CKIP-1-28 ({Delta}) clones were measured at the indicated days after tumor inoculation. n = 7.

 
To examine the effect of CKIP-1 expression on in vivo tumor growth, we used a mouse tumor xenograft model. The parent or stable transfectants were s.c. injected into the right flank of BALB/c-nu/nu (nude) mice. Eight days after injection, the tumor volume of the stable CKIP-1 transfectants was 50% to 70% smaller than that of the parent or mock transfectants (Fig. 6C and D). The in vivo tumor growth of the stable CKIP-1 transfectants was attenuated throughout the observation period (Fig. 6D, right). We resected the tumor and examined CKIP-1 expression and Akt phosphorylation level. In vivo growth of stable CKIP-1 transfectants did not affect the HA-tagged CKIP-1 expression level (Fig. 6D, left). In these tumors, Akt phosphorylation was decreased compared with the tumors of parent or stable mock transfectants (Fig. 6D, left). These results suggested that CKIP-1 decreases tumorigenicity in vivo by suppressing Akt activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that Akt interacts with its substrates and regulates cell survival and proliferation functions. It was also reported recently that some molecules, which are not Akt substrates, bind to Akt and regulate its activity and function (13, 25–27). Among them, COOH-terminal modulator protein (25) and PH domain leucine-rich repeat protein phosphatase (26) were reported as negative regulators of Akt. Fused toes protein-1 was reported as the positive regulator of Akt (27). These proteins bound to the COOH-terminal regulatory domain of Akt. On the other hand, TCL1, a positive regulator of Akt, formed a complex via the PH domain of Akt (13). In this study, we searched another regulatory proteins of Akt.

We identified CKIP-1 as a novel Akt-binding protein using an E. coli–based two-hybrid system. This interaction was reproducible in vitro and in mammalian cells (Fig. 1). CKIP-1 interacted with K179A-Akt, K179A/T308A/S473A-Akt, or T308D/S473D-Akt as well as wt-Akt (data not shown). Endogenous CKIP-1 and Akt formed the complex under the conditions of serum starvation, insulin-like growth factor-I, or epidermal growth factor stimulation (data not shown). These results indicate that CKIP-1 forms a complex with Akt regardless of Akt phosphorylation status and activity. CKIP-1 expression was decreased in tumor tissues compared with that in normal tissues (Supplementary Fig. S3). CKIP-1 expression was inversely correlated with Akt phosphorylation (Supplementary Fig. S4). Thus, the expression level of endogenous CKIP-1 might be one of the determinants of steady-state activity of Akt in cells. CKIP-1 contains sequences 83-KSKSRS-88, 222-RRRADS-227, and 266-KGRCAS-271, which were similar to Akt phosphorylation sites. The phosphorylated Akt substrate antibody did not detect phosphorylated forms of CKIP-1 protein even when CKIP-1 was coexpressed with Akt in cells (data not shown). Furthermore, either phosphorylation-deficient S88A/S227A/S271A-CKIP-1 or phosphorylation-mimic S88D/S227E/S271D-CKIP-1 behaved like wt-CKIP-1 in the interaction with Akt and in the suppression of Akt phosphorylation (data not shown). Therefore, CKIP-1 might not be an Akt substrate. CKIP-1 bound to the PH domain of Akt (Fig. 1B), and its expression decreased the phosphorylated Akt levels (Fig. 2). The PH domain is the binding domain to phosphoinositides. In the case of Akt, the translocation at the plasma membrane and the conformational change accompanied by the interaction between the Akt PH domain and phosphoinositides are critical for its activation (21–23). The PH domain is also associated with binding to proteins (10). It has been reported that the PH domain of Akt is associated with TCL1 (11, 12), myosin II (28), inosine-5' monophosphate dehydrogenase (29), and c-Jun NH2-terminal kinase–interacting protein 1 (30). Among them, TCL1 was reported to promote Akt kinase activity. The dimerization of TCL1 promotes the autophosphorylation of Akt at Ser473 (13). Thus, TCL1 is a positive regulator of Akt. However, TCL1 expression is limited in embryos, germ cells, and lymphocytes (13) compared with the ubiquitous expression of Akt (31). In contrast to TCL1, CKIP-1 was ubiquitously expressed in various tissues (14). Moreover, we found an inverse correlation between CKIP-1 expression and Akt phosphorylation (Supplementary Fig. S4). These results suggest that CKIP-1 serves as a negative regulator of Akt in various tissues.

CKIP-1 interacted with Akt via its NH2-terminal region that contained the PH domain (Fig. 3). It has been reported that the PH domain of CKIP-1 binds to phosphoinositides (15, 16). However, these reports do not agree on the variety of phospholipids bound to CKIP-1. The reason is that the condition of lipids in the lipid overlay assay is different from the in vivo condition. In our hands, CKIP-1 bound to a broad spectrum of phospholipids in ELISA and in the lipid overlay assay (data not shown). Using a cosedimentation assay, which is closer to the in vivo condition, we estimated the phospholipid-binding ability of CKIP-1. We discovered that CKIP-1 preferentially bound to PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 (Fig. 5). It is well known that Akt binds to PtdIns(3,4,5)P3, and this interaction causes Akt activation (22, 23). Therefore, it is possible that CKIP-1 competes with and inhibits Akt binding to PtdIns(3,4,5)P3. To estimate the possibility, we generated a CKIP-1 mutant, KRW-CKIP-1, which lacked both lipid-binding ability and plasma membrane–localizing activity (15). Contrary to our expectations, KRW-CKIP-1 also down-regulated Akt phosphorylation level (Fig. 5). We found that another CKIP-1 PH domain mutant, which also disrupted lipid-binding ability, suppressed endogenous Akt phosphorylation under heat shock stimulation (data not shown). Moreover, CKIP-1 suppressed phosphorylation and kinase activity of active Akt mutant, E40K-Akt (Fig. 2B). These results indicate that the suppression of Akt activity by CKIP-1 is not due to competition with Akt for phosphoinositide binding.

CKIP-1 was reported to dimerize through its LZ motif (16). However, the significance of this dimerization is unclear. We show herein that the LZ motif of CKIP-1 played an important role in suppressing Akt activity. The {Delta}C-CKIP-1 mutants and 200-343-CKIP-1 mutant, lacking the LZ motif, did not modulate Akt phosphorylation status (Figs. 3 and 4). Conversely, the {Delta}N199-CKIP-1, containing the LZ motif, increased Akt kinase activity without binding to it (Fig. 3). Because {Delta}N199-CKIP-1 could bind to wt-CKIP-1 (Fig. 4) and competitively inhibited wt-CKIP-1 binding to Akt (Fig. 4D), expression of {Delta}N199-CKIP-1 might elevate Akt activity by sequestering endogenous wt-CKIP-1 from Akt. These results suggest that binding to Akt via its NH2 terminus and binding to CKIP-1 via its LZ motif are both required for CKIP-1–mediated Akt inactivation. Because CKIP-1 expression also suppressed FKHRL1 phosphorylation by active E40K-Akt (Fig. 2B), CKIP-1 dimerization via LZ region might result in inhibition of Akt signaling by masking substrate recognition domain in Akt. To confirm the results, we tried to knock down endogenous CKIP-1 expression using >10 small interfering RNAs from different companies. Unfortunately, we were unsuccessful in several cell lines. Further study is needed to confirm the role of endogenous CKIP-1 expression in the regulation of Akt kinase activities.

It has been reported that Akt facilitates cell cycle progression and cell proliferation by phosphorylating and inactivating CDK inhibitors, such as p21Waf1/Cip1 and p27Kip1 (19, 20, 24). Akt also phosphorylates GSK-3ß and blocks its kinase activity, thereby allowing cyclin D1 to accumulate (32). We showed that the growth of stable CKIP-1 transfectants was slower than that of parent or mock transfectants in vitro and in vivo (Fig. 6). These stable CKIP-1 transfectants exhibited impaired Akt signaling. Additionally, transient and stable expression of CKIP-1 increased endogenous p27Kip1 protein level (Fig. 6; Supplementary Fig. S2). It is also known that the Ras-mitogen-activated protein kinase (MAPK) pathway is a major pathway for cell proliferation. However, MEK/extracellular signal-regulated kinase kinase or MAPK phosphorylation levels were not changed (data not shown). Moreover, growth retardation in stable CKIP-1 transfectants was rescued by E40K-Akt expression (Supplementary Fig. S5). These results suggest that suppressing endogenous Akt activity by CKIP-1 expression results in tumor cell growth attenuation.

It has been reported that the PI3K-Akt pathway is frequently activated in cancer. The chromosomal deletion or loss of functional mutation of PTEN has been detected in many cancers. The gene encoding p110{alpha} (PIK3CA) amplification or active mutation also occurred in several cancer cell lines. The amplification of the akt gene occurred in some cases of ovarian, breast, and pancreatic cancer (2). Therefore, the PI3K-Akt pathway is being seen as a molecular cancer therapeutic target. Previous studies suggest that some anticancer drugs have targeted the PI3K-Akt pathway (1, 2). It was reported that LY294002 treatment or dominant-negative Akt expression enhances the sensitivity of Akt-activated cancer cell lines to anticancer drugs (33, 34). In our experiments, no apoptotic change could be detected by expressing CKIP-1 alone. However, the numbers of cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 fragments increased after etoposide treatment in CKIP-1–stable transfectants compared with parent or mock-stable transfectants (data not shown). Akt phosphorylation increased in parent or mock cells after drug treatment, whereas it was suppressed in stable CKIP-1 transfectants. Moreover, the sub-G1 fraction increased after 48 h of etoposide treatment in stable CKIP-1 transfectants (data not shown). These results suggest that CKIP-1 enhances the sensitivity to anticancer drugs by suppressing Akt activity.

It is thought to be difficult to generate an Akt-specific inhibitor because its kinase domain has high homology with other AGC kinases. Akt-specific inhibitors with novel mechanisms of action are currently under development (1, 2, 35). These inhibitors are specifically targeted to the Akt PH domain, including perifosine and SH-5 (36, 37). They prevent Akt membrane localization by interacting with the PH domain. Because CKIP-1 targets the Akt PH domain and suppresses Akt kinase activity, this novel suppressing mechanism of Akt is an intriguing therapeutic target.


   Acknowledgments
 
Grant support: Ministry of Education, Culture, Sports, Science, and Technology of Japan Grant-in-Aid for Scientific Research on Priority Area "Cancer" 17016012 (T. Tsuruo) and 18015008 (N. Fujita); Ministry of Education, Culture, Sports, Science and Technology of Japan National Project on Protein Structural and Functional Analyses (N. Fujita); Araki Memorial Foundation for Medical and Biochemical Researches (N. Fujita); and Vehicle Racing Commemorative Foundation (N. Fujita).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

4 http://www.ncbi.nlm.nih.gov/BLAST/ Back

Received 3/20/07. Revised 6/27/07. Accepted 7/12/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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