C-NAP1, A NOVEL CENTROSOMAL COILED-COIL PROTEIN AND CANDIDATE SUBSTRATE OF THE CELL-CYCLE-REGULATED PROTEIN-KINASE NEK2

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle-regulated k inase structurally related to the mitotic regulator NIMA of Aspergillu s nidulans. In human cells, Nek2 associates with centrosomes, and over expression of active Nek2 has drastic consequences for centrosome Stru cture. Here, we describe the molecular characterization of a novel hum an centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protei n 1), first identified as a Nek2-interacting protein in a. yeast two-h ybrid screen. Antibodies raised against re; combinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectr on microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles, On Western blot s, anti-C-Nap1 antibodies recognized a large protein (>250 kD) that wa s highly enriched in centrosome preparations, Sequencing of overlappin g cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD a nd comprises extended domains of predicted coiled-coil structure. Wher eas C-Nap1 was concentrated at centrosomes in all interphase cells, im munoreactivity at mitotic spindle poles was strongly diminished. Final ly, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins i n vivo. Based on these findings, we propose a model implicating both N ek2 and C-Nap1 in the regulation of centriole-centriole cohesion durin g the cell cycle.