Cloning, expression, and promoter structure of a mammalian inner centromere protein (INCENP)

The term `chromosomal passenger proteins' was first coined to describe a group of cellular proteins that tightly associate with chromosomes during the early stages of mitosis, localize at the centromere at metaphase, and subsequently relocate to other subcellular regions at the commencement of anaphase (Earnshaw and Bernat 1990). One of these proteins, the Inner Centromere Protein or INCENP, was first identified using antibodies raised against chicken chromosomal scaffold proteins (Cooke et al. 1987). Like other passenger proteins, chicken INCENP abruptly dissociates from the chromosomes at the metaphase/anaphase transition and associates with the overlapping microtubules of the central spindle. In addition, a subpopulation of protein concentrates in the presumptive cleavage furrow prior to the commencement of furrowing, suggesting a crucial role for INCENP in cytokinesis (Earnshaw and Cooke 1991). Chicken cells contain two INCENP proteins (designated class I and II) of 133 and 145 kDa respectively. Molecular analysis has revealed that both classes arise from a single, differentially spliced primary transcript with class II, differing from class I by the insertion of 114 additional nucleotides (Mackay et al. 1993). Chicken INCENPs are highly basic proteins containing long coiled-coil domains, five putative nuclear localization sites, and many putative phosphorylation sites. They share little homology to other known proteins (Mackay et al. 1993). Both chicken INCENP cDNAs have been expressed in mammalian cells and shown to localize identically to the endogenous mammalian INCENP protein, suggesting conservation of nuclear, chromosomal, spindle, and cell cortex factors that interact with INCENP during the cell cycle (Mackay et al. 1993). Expression of an amino terminal mutant of chicken INCENP (INCENP sub(43-839)) in mammalian cells has identified an essential role for the first 42 residues in centromere targeting and transfer of the protein from chromosomes to the spindle and cell cortex during the metaphase/anaphase transition (Mackay et al. 1993, 1998). Deletion of the coiled-coil domain prevented microtubule association, but had no effect on spindle association (Mackay et al. 1993). A truncation derivative (INCENP sub(1-405)) that targets correctly to centromeres does not redistribute at anaphase and acts as a dominant-negative mutant, disrupting both metaphase chromosome alignment and cytokinesis in mammalian cells (Mackay et al. 1998). Recently, a Xenop