Proteomic analysis of apoptosis related proteins regulated by proto-oncogene protein DEK.
A nuclear phosphoprotein, DEK, is implicated in certain human diseases, such as leukemia and antoimmune disorders, and a major component of metazoan chromatin. Basically as a modulator of chromatin structure, it can involve in various DNA and RNA-dependent processes and function as either an activator or repressor. Despite of numerous efforts to suggest the biological role of DEK, direct target proteins of DEK in different physiological status remains elusive. To investigate if DEK protein triggers the changes in certain protein networks, DEK was knocked down at both types of cell clones using siRNA expression. Here we provide a catalogue of proteome profiles in total cell lysates derived from normal HeLa and DEK knock-down HeLa cells and a good in vitro model system for dissecting the protein networks due to this proto-oncogenic DEK protein. In this biological context, we compared total proteome changes by the combined methods of two-dimensional gel electrophoresis, quantitative image analysis and MALDI-TOF MS analysis. There were a large number of targets for DEK, which were differentially expressed in DEK knock-down cells and consisted of 58 proteins (41 up-regulated and 17 down-regulated) differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. In the identified 58 spots, 16% of proteins are known to be associated with apoptosis. Among others, we identified apoptosis related proteins such as Annexins, Enolase1, Lamin A, and Glutathione-S-transferase omega 1. These results are consistent with recent studies indicating the crucial role of DEK in apoptosis pathway. We further demonstrated by ChIP analysis that knock-down of DEK caused hyperacetylation of histones around Prx VI promoter which is upregulated in our profile. Using immunoblotting analysis, we have demonstrated the modulation of other caspase-dependent apoptosis related proteins by DEK knock-down and further implicate its role in apoptosis pathway.
Kim
Dong-Wook
DW
Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, South Korea.
Chae
Jung-Il
JI
Kim
Ji-Young
JY
Pak
Jhang Ho
JH
Koo
Deog-Bon
DB
Bahk
Young Yil
YY
Seo
Sang-Beom
SB
D000107:Acetylation; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D054465:Peroxiredoxin VI / Q000235:genetics / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011401:Promoter Regions, Genetic; D020543:Proteome / Q000032:analysis*; D000090063:Proto-Oncogene Mas; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002868:Chromosomal Proteins, Non-Histone; C084738:DEK protein, human; D006657:Histones; C000715470:MAS1 protein, human; D015513:Oncogene Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D020543:Proteome; D000090063:Proto-Oncogene Mas; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI