RESUMEN
Two thousand sixty-eight multi-purpose expression clones for the 326 candidate genes related to gastric or liver cancers were constructed using the Gateway system. These clones can be expressed as His, Glutathione-S-transferase (GST) or Enhanced version of the green fluorescent protein (EGFP) fusion proteins in E. coli, insect cells or mammalian cells. For the 246 E. coli expression clones, the GST fusion proteins had greater expression efficiency and solubility than the His fusion proteins. Approximately 20% of the expressed proteins had unexpected molecular weights. A detailed sequence analysis of these clones revealed frameshift mutations resulting from insertion, deletion or substitution of nucleotides. The results indicate that these changes in the candidate genes may affect the occurrence of gastric or liver cancers. In addition, when 105 proteins, which were expressed in E. coli at very low or undetectable levels, were expressed in insect cells, 76% of the proteins were expressed very well and most were soluble. We also found that most of the 30 proteins prepared using EGFP mammalian expression clones were localized to cellular compartments expected by Gene ontology (GO) and this localization was unaffected if the EGFP-fusion was at the N-terminal or C-terminal region of the protein. Antibody production and subcellular localization analysis of the candidate genes as well as a screen of genes involved in carcinogenesis pathways are currently in progress using these expression clones. These studies provide a valuable resource for developing a better understanding of the molecular mechanism of carcinogenesis in both gastric and liver cancer and would be very helpful in diagnosis and therapeutic predictions.
Asunto(s)
Clonación Molecular , Neoplasias Hepáticas/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Neoplasias Gástricas/metabolismo , Animales , Línea Celular , Células Cultivadas , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/metabolismo , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Histidina/metabolismo , Humanos , Riñón/citología , Neoplasias Hepáticas/genética , Ratones , Células 3T3 NIH , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Spodoptera/citología , Spodoptera/genética , Spodoptera/metabolismo , Neoplasias Gástricas/genética , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
CKII plays a significant role in cell proliferation and cell cycle control. In this report, yeast two-hybrid assay and pull-down assay demonstrate that CKBBP2/CRIF1 associates with the beta subunit of CKII in vitro and in vivo. Recombinant CKBBP2/CRIF1 is phosphorylated in vitro by purified CKII and by CKII inhibitor apigenin-sensitive protein kinase in HEK293 cell extract. Phosphoamino acid analysis and mutational analysis indicate that CKII phosphorylates serine at residue 221 within CKBBP2/CRIF1. Furthermore, serine to alanine mutation at residue 221 abrogates the phosphorylation of CKBBP2/CRIF1 observed in HEK293 cell extract, indicating that CKII is a major kinase that is responsible for phosphorylation of CKBBP2/CRIF1. As compared with the wild-type CKBBP2/CRIF1 or nonphosphorylatable mutant CKBBP2(S221A) (in which the serine-221 is replaced by alanine), overexpression of CKBBP2(S221E) in COS7 cells promotes cell proliferation. Taken together, the present results suggest that CKII may be involved in cell proliferation, at least in part, through the phosphorylation of serine-221 within CKBBP2/CRIF1.