DDIT3/CHOP and the sarcoma fusion oncoprotein FUS-DDIT3/TLS-CHOP bind cyclin-dependent kinase 2
© Bento et al; licensee BioMed Central Ltd. 2009
Received: 1 July 2009
Accepted: 17 December 2009
Published: 17 December 2009
The DDIT3 gene encodes a transcription factor belonging to the CCAAT/enhancer binding protein (C/EBP) family. It is normally expressed at very low levels but is activated by cellular stress conditions and induces G1 arrest and, in some cell types, apoptosis. DDIT3 is found as a part of the fusion oncogene FUS-DDIT3 that is causal for the development of myxoid/round-cell liposarcomas (MLS/RCLS).
In the present study, we searched for putative interaction partners of DDIT3 and the oncogenic FUS-DDIT3 among G1 cyclins and cyclin-dependent kinases. We found that FUS-DDIT3 and the normal DDIT3 bind CDK2. In addition, CDK2 showed an increased affinity for cytoskeletal proteins in cells expressing FUS-DDIT3 and DDIT3.
We conclude that DDIT3 binds CDK2 and that many of the observed biological effects of DDIT3 may involve interaction with CDK2.
DDIT3 (GADD153, CHOP) encodes a transcription factor belonging to the CCAAT/enhancer binding protein (C/EBP) family . DDIT3 is normally transcribed at very low levels but is elevated upon DNA damage in cellular stress conditions [2–6]. The DDIT3 protein has a central role in endoplasmatic reticulum stress and DNA damage response by inducing cell cycle arrest and apoptosis [7, 8]. DDIT3 has recently been implicated in the stress response leading to death of pancreatic insulin producing β-cells  and it may also be a part of cellular stress conditions causing neurodegenerative disorders . In addition, DDIT3 is believed to be involved in growth cessation and terminal differentiation of lipoblasts, osteoblasts and erythrocytes [11–13]. DDIT3 forms heterodimers with several other C/EBP family members  as well as other leucine zipper carrying proteins [15, 16] and the heterodimers are believed to act as dominant negative inhibitors of transcription .
DDIT3 is also a part of a fusion oncogene critical for the development of myxoid/round cell liposarcoma (MLS/RCLS) [17, 18]. The tumor cells carry the chromosomal translocation t(12;16)(q13;p11) that results in fusion of DDIT3 to FUS (also called TLS) or more rarely the t(12;22)(q13;q12) that fuses DDIT3 to EWSR1 [19–21]. The chimerical FUS-DDIT3 oncoprotein functions as an abnormal transcription factor  and localizes to distinct nuclear structures in cultured cells [23, 24]. MLS/RCLS cells exhibit abnormal expression profiles of cell cycle controlling factors and among them cyclins and cyclin-dependent kinases (CDKs) . In addition, the DDIT3-binding C/EBPα has been shown to interact and inhibit the kinase activity of CDK2 and CDK4 . Based on these observations, we searched for putative interaction partners of the oncogenic FUS-DDIT3 with G1 cyclins and CDKs as these might provide insight into the molecular mechanisms by which the chimerical oncoprotein induces malignancy.
CDK2 and cyclin E colocalize with FUS-DDIT3
FUS-DDIT3 binds CDK2 through its DDIT3 part
CDK2 expression, phosphorylation status and turnover is not changed by FUS-DDIT3
CDK2 protein binding affinity is altered in FUS-DDIT3 and DDIT3 expressing cells
In the present study, we used immunofluorescence microscopy to investigate putative colocalization between FUS-DDIT3 and cyclins/cyclin-dependent kinases involved in G1 cell cycle control. CDK2 and the CDK2 binding cyclin E were found to change their localization in FUS-DDIT3 expressing cells to a pattern that was identical to the FUS-DDIT3 nuclear granules reported earlier . This suggests that CDK2 and cyclin E are translocated to nuclear structures defined by the FUS-DDIT3 protein. As the DDIT3 part is considered a DNA-binding transcription factor , we speculate that the FUS-DDIT3 defined structures may contain active chromatin and mRNA at different stages of processing. Thus, the accumulation of CDK2 and cyclin E to such nuclear regions may result in changed phosphorylation patterns and regulation of substrates present at these foci.
DDIT3 is able to form DNA-binding heterodimers with C/EBPα through its leucine zipper region  and C/EBPα is reported to bind CDK2 and inhibit its kinase activity . Therefore, it is possible that the binding between DDIT3 and CDK2 is mediated through C/EBPα or another C/EBP protein. However, we here show that a DDIT3 mutant lacking the leucine zipper domain binds CDK2, which implies a binding between DDIT3 and CDK2 that is independent of other C/EBP proteins. DDIT3 and C/EBPα contain several regions with sequence similarities but the proline-histidine rich part reported to bind CDK2 in C/EBPα  (Figure 2c) is not present in DDIT3. This suggests that DDIT3 binds CDK2 through a different mechanism than C/EBPα.
Analyses of GFP-immunoprecipitates failed to detect cyclin E in these samples. The absence of cyclin E in CDK2 immunoprecipitates bears resemblance to previous reports showing that C/EBPα disrupts CDK2/cyclin complexes leading to growth arrest . Hence, it is possible that the DDIT3 bound to CDK2 disrupts CDK2/cyclin E complexes in similar way as C/EBPα. Consequently, since cyclin E is a major regulator of CDK2 kinase activity in G1, the DDIT3 binding may alter CDK2 activity in DDIT3 and FUS-DDIT3 expressing cells. We did however not detect a change in phosphorylation status of CDK2 in FUS-DDIT3 expressing cells compared to control cells. To further analyze the functional effects of DDIT3/FUS-DDIT3 binding to CDK2, we immunoprecipitated CDK2 in cells transiently transfected with FUS-DDIT3, DDIT3 and GFP constructs. Analysis of the precipitates revealed enhanced binding of CDK2 to components of the cytoskeleton in cells expressing FUS-DDIT3 and DDIT3. An increased affinity for cytoskeletal components and crosstalk between cell cycle proteins and cytoskeletal regulatory proteins could lead to changes in cytoskeleton structure .
In conclusion, we show that CDK2 is translocated to nuclear structures defined by the FUS-DDIT3 oncoprotein and that it binds the DDIT3 part of the chimera. Cyclin E is also recruited to FUS-DDIT3 nuclear structures but can not be found in CDK2-containing DDIT3/FUS-DDIT3 precipitates. The interaction of FUS-DDIT3 and DDIT3 with CDK2 appears to alter the binding affinity of CDK2, possibly leading to changed phosphorylation patterns and regulation of cytoskeletal or other proteins. Many of the observed biological effects of DDIT3 may involve the interaction with CDK2.
Cell culture and transfection
Human HT1080 fibrosarcoma cells were grown in RPMI 1640 medium (Sigma-Aldrich) supplied with 10% fetal bovine serum, penicillin (50 U/ml) and streptomycin (50 μg/ml) at 37°C in 5% CO2. Cells were transiently transfected at 50-70% confluence using the FuGENE 6 transfection reagent (Roche) with a 3:1 FuGENE:DNA ratio, according to the instructions supplied by the manufacturer.
Cells were fixed 24 hours after transfection in 3.7% formaldehyde in PBS for 10 min, rinsed in PBS and stained with rabbit polyclonal antibodies for CDK2 (C5223, Sigma-Aldrich), CDK4 (C8218, Sigma-Aldrich), cyclin D1 (M7155, Dako) or cyclin E (C4976, Sigma-Aldrich) that were detected using Cy3 conjugated secondary antibodies (Fluorolink, Amersham Biosciences). Finally, slides were mounted using DAPI/DABCO solution (Sigma-Aldrich), incubated over night at room temperature and then imaged using a Zeiss LSM510 META confocal microscope system.
For cloning of CDK2 the following primers were used: CDK2 forward GATCTCGAGCCACCATGGAGAACTTCCAAAAG, CDK2 reverse CAAGGATCCCGGAGTCGAAGATGGGGTACT. The CDK2 fragment was cloned into the pDsRed1-N1 vector (Clontech Laboratories) in frame with the DsRed1 sequence. All constructs were sequenced to exclude mutant clones. FUS-DDIT3-GFP, DDIT3-GFP and FUSA-GFP were previously described [24, 29]. The FUS-DDIT3ΔLZ-GFP construct, expressing a protein lacking the 38 most C-terminal amino acids (including the leucine zipper region), is described elsewhere . Stably transfected FUS-DDIT3 cells has been described previously .
HT1080 cells were co-transfected with CDK2-DsRed and/or FUS-DDIT3-GFP/FUS-DDIT3-ΔLZ/DDIT3-GFP/FUSA-GFP/GFP constructs at 50-70% confluence. Twenty-four hours post transfection, cells were washed in PBS, scraped and lysed in NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0) containing protease inhibitor (Complete Mini, Roche). Debris was removed by centrifugation at 14,000 g and supernatants were used for co-immunoprecipitations. Two microgram of antibodies specific for GFP (8372-2, BD Pharmingen) or CDK2 (C5223, Sigma-Aldrich) were prebound to protein A agarose beads (Millipore) blocked with 5% bovine serum albumin in NP-40 buffer overnight. Cell lysates were incubated with antibody-beads mixtures for 4 h at room temperature. Beads were washed 5 times with NP-40 buffer and proteins were released from beads by addition of 4× NuPAGE LDS sample buffer (Invitrogen) and incubation at 95°C. Supernatants were subsequently analyzed with western blot or with SYPRO Ruby gel staining (Sigma-Aldrich) according to the instructions supplied by the manufacturer. SYPRO stained gels were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) at the Proteomics Core Facility at University of Gothenburg. For experiments with phospho-CDK2 antibodies (see below), transfected cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% Sodium deoxycholate, 0.1% SDS) containing protease inhibitors and phosphatase inhibitors (NaF 10 mM, Na3VO4 1 mM).
Western Blot Analysis
Proteins were separated using NuPAGE 4-12% Bis-Tris gels (Invitrogen), blotted onto PVDF membranes (Millipore) and probed with the following primary antibodies: CDK2 (sc-6248, Santa Cruz Biotechnology), p-CDK2 Thr14/Tyr15 (Santa Cruz Biotechnology), p-CDK2 Thr160 (Cell Signaling), cyclin E (C4976, Sigma-Aldrich), β-actin (mAbcam8226, Abcam) and GFP (8371-2, BD Pharmingen). Primary antibodies were detected with alkaline phosphatase conjugated secondary antibodies (Dako) and bands were visualized with BCIP/NBT Alkaline Phosphatase Substrate (Sigma-Aldrich).
The amino acid sequences of DDIT3 and C/EBPα were aligned using the ClustalW algorithm available at the Universal protein resource (UniProt) .
cyclin-dependent kinase 2
CCAAT/enhancer binding protein
C/EBP homologous protein
DNA damage-inducible transcript 3
green fluorescent protein
liquid chromatography mass spectrometry
translocated in liposarcoma.
We thank Anita Olofsson and Farzaneh Alemirad for technical assistance. We acknowledge the Proteomics Core Facility at University of Gothenburg for mass spectrometric analyses. This work was supported by grants from the Swedish Cancer Society, Assar Gabrielssons Research Foundation and the Johan Jansson Foundation for Cancer Research.
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