DDIT3/CHOP and the sarcoma fusion oncoprotein FUS-DDIT3/TLS-CHOP bind cyclin-dependent kinase 2

Abstract

Background: 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).

Results: 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.

Conclusions: We conclude that DDIT3 binds CDK2 and that many of the observed biological effects of DDIT3 may involve interaction with CDK2.

Figure 1

CDK2 and cyclin E colocalize with FUS-DDIT3 in transiently transfected HT1080 cells. The endogenous distribution of CDK2 and cyclin E seen in red is detected by antiserum specific for these proteins. Ectopically expressed GFP-tagged FUS-DDIT3 is shown in green. The DAPI dye is used to stain nuclei blue. CDK4 was not seen to accumulate in FUS-DDIT3-containing granules and no granules were formed in cells overexpressing the GFP protein only. Bars indicate 5 μm.

Figure 2

Co-immunoprecipitation of DDIT3 and CDK2. (a) HT1080 cells were co-transfected with the indicated GFP constructs and CDK2-DsRed1. Proteins in cell lysates were further immunoprecipitated with anti GFP serum and recombinant CDK2-DsRed1 protein was detected by western blot using CDK2 antibodies. Input shows proteins before immunoprecipitation of cell lysates while IP shows proteins present in immunoprecipitates. (b) HT1080 cells were transiently transfected with the indicated GFP constructs and cellular proteins were immunoprecipitated with CDK2 antibodies. Recombinant proteins in samples were detected with anti GFP serum. The most intense bands in the input show the correct size of the GFP-tagged protein products while additional unspecific bands were also reacting with the GFP antisera in these samples. (c) ClustalW alignment of DDIT3 and C/EBPα amino acid sequences. A region shown to bind CDK2 in C/EBPα is boxed. Letters indicate standard amino acid abbreviations. Amino acids with related physicochemical characteristics are displayed below the sequences as: '*' (identical residue), ':' (highly similar residue), '.' (weakly similar residue). The leucine zipper region is seen towards the C-terminal in both proteins.

Figure 3

Expression and phosphorylation status of CDK2. Western blot analysis of cell lysates from HT1080 cells transiently transfected with FUS-DDIT3-GFP and GFP. β-actin is used as loading control.

Figure 4

SYPRO stained gel of co-immunoprecipitated proteins in extracts from HT1080 cells transiently transfected with either of DDIT3-GFP, FUS-DDIT3-GFP or GFP constructs. CDK2 antibodies were used for immunoprecipitations. Boxes indicate regions of the gel analyzed by mass spectrometry. Three proteins were enriched in immunoprecipitates from DDIT3 and FUS-DDIT3 expressing cells (region 1: plectin, region 2: myosin, region 3: vimentin). Arrows indicate the differentially immunoprecipitated vimentin bands present in lanes of DDIT3 and FUS-DDIT3 cells but absent in that of GFP cells. These bands migrate slightly slower than the thicker bands inside the same region. The thicker bands were identified as rabbit heavy chain immunoglobulin molecules and these originate from the antibodies used for immunoprecipitation of CDK2 and are present in all three lanes.