Abstract
KM-HN-1 is a C-terminal coiled-coil domain containing protein previously referred to as image clone MGC33607. This protein has been previously identified as a cancer/testis antigen and reported as nuclear and chromatin localizing protein. We raised polyclonal antisera with the GST fusion protein and identified them as a 105 kDa protein. Motif analysis showed that this protein harbors the leucine zipper motif in internal 1/3 region and the coiled-coil domain in the C-terminal region. Using the full length and various deletion mutants, we determined the motif that governs the subcellular localization of KM-HN-1. Immunofluorescence staining of the endogenous KM-HN-1 and various kinds of GFP-tagged KM-HN-1 revealed that KM-HN-1 localizes to the centrosomes as well as nucleus. The centrosomal localization-determining region of this protein is C-terminal coiled-coil domain in which the leucine zipper motif and the nuclear export signal (NES) harbor.
Highlights
KM-HN-1 (MGC33607) was identified as a novel human cancer/testis antigen, in an attempts to discover cancer-specific target genes for cancer therapeutics development
Cdc14 plays a key role in the mitotic exit network and the centrosome cycle, from budding yeasts to metazoans (Pereira and Schiebel, 2001; Trautmann and McCollum, 2002; Stegmeier and Amon, 2004). hCdc14A performs a crucial function in centrosome duplication and the completion of mitosis in human cells (Kaiser et al, 2002; Mailand et al, 2002)
KM-HN-1 has previously been identified as a cancer/testis antigen, the study of KM-HN-1 has mainly been limited to mRNA expression level analyses using several cancer tissues and cell lines (Monji et al, 2004; Condomines et al, 2007)
Summary
KM-HN-1 (MGC33607) was identified as a novel human cancer/testis antigen, in an attempts to discover cancer-specific target genes for cancer therapeutics development. We have obtained a KM-HN-1 clone from a yeast two-hybrid screening, using hCdc14A as bait. HCdc14A performs a crucial function in centrosome duplication and the completion of mitosis in human cells (Kaiser et al, 2002; Mailand et al, 2002). The SMC motif originates from SMC protein, which performs pivotal function during chromosome segregation (Cobbe and Heck, 2000). In addition to the coiled-coil domain KM-HN-1 harbors a bipartite NLS (nuclear localization signal) (Boulikas, 1993) followed by a leucine zipper region, in the internal 1/3 region (Figure 1). The leucine zipper motif functions as a protein dimerization domain of a variety of DNA binding proteins (Landschulz et al, 1988). The clones initially obtained evidenced centrosomal localization and colocalization with hCdc14A in the centrosome. We determined the region of KM-HN-1 critical to centrosomal localization, and other features of the intracellular localizations using KM-HN-1 deletion mutants
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