Initial characterization of the CDP2Cux2 protein

Abstract

The CDP1 (CCAAT-d isplacement protein 1) and cux1 (Cut homeobox1 ) genes were originally identified as the human and mouse orthologs of Drosophila melanogaster cut.To begin to characterize the CDP2/Cux2 protein, I generated a number of reagents to analyze its expression and activity. Polyclonal antibodies were obtained by injecting rabbits with glutathione-S-transferase fusion proteins containing antigenic regions of the Cux2 protein. The antibodies were then characterized in Western blot analysis and electrophoretic mobility shift assays (EMSA). In total, 5 antibodies were produced against different regions of Cux2. These antibodies were able to specifically recognize CDP2 and Cux2 but not CDP1 or Cux1. Expression of the Cux2 protein was found in only one among 19 neuronal cell lines: the SH-SY5Y human neuroblastoma cell line. Histidine-tagged fusion protein containing various combinations of Cut repeats and Cut homeodomain were generated to investigate the DNA binding properties of CDP2/Cux2. The CR1CR2, CR2CR3HD and CR3HD domains were found to exhibit similar DNA binding specificities as the corresponding domains of CDP1/Cux2, however, analysis of DNA binding kinetics revealed that all of these combinations of domains made rapid but transient interactions with DNA. Mammalian expression vectors were engineered with epitope tags at the N- and C-termini of CDP2/Cux2. The full-length protein was found to localize in the nucleus and also to make a rapid but transient interaction with DNA. In contrast to CDP1/Cux1, the CDP2/Cux2 protein did not appear to be subject to specific proteolytic processing