In a previous study, we demonstrated that the forkhead associated (FHA) domain of pKi-67 interacts with the novel kinesin-like protein, Hklp2 (Sueishi, M., Takagi, M., and Yoneda, Y. (2000) J. Biol. Chem. 275, 28888 -28892). In this study, we report on the identification of a putative RNA-binding protein of 293 residues as another binding partner of the FHA domain of pKi-67 (referred to as NIFK for nucleolar protein interacting with the FHA domain of pKi-67). Human NIFK (hNIFK) interacted with the FHA domain of pKi-67 (Ki-FHA) efficiently in vitro when hNIFK was derived from mitotically arrested cells. In addition, a moiety of hNIFK was co-localized with pKi-67 at the peripheral region of mitotic chromosomes. The hNIFK domain that interacts with Ki-FHA was mapped in the yeast two-hybrid system to a portion encompassed by residues 226 -269. In a binding assay utilizing Xenopus egg extracts, it was found that the mitosis-specific environment and two threonine residues within this portion of hNIFK (Thr-234 and Thr-238) were crucial for the efficient interaction of hNIFK and Ki-FHA, suggesting that hNIFK interacts with Ki-FHA in a mitosis-specific and phosphorylation-dependent manner. These findings provide a new clue to our understanding of the cellular function of pKi-67.The Ki-67 antigen (pKi-67), originally identified as the antigen for a monoclonal antibody raised against the nuclear extract from a Hodgkin's lymphoma-derived cell line, was characterized as a class of proteins that localize around mitotic chromosomes (1). As a result, it is assumed that pKi-67 is involved in mitotic chromosome organization. pKi-67 is a convenient cell proliferation marker, since its expression is restricted to growing cells (2). Although the recent identification and characterization of a marsupial counterpart of pKi-67, which is referred to as chmadrin, suggests that pKi-67 plays some type of role in the organization of higher order chromatin structure (3), the actual role of pKi-67 in the cell cycle progression remains unclear.The N-terminal portion of pKi-67 is well conserved between human pKi-67 and chmadrin (62% identical) and contains a forkhead associated (FHA) 1 domain. It was originally reported that the FHA domain constituted a region that has been conserved in a subset of forkhead-type transcription factors (4). The sequence profile has been reported for a variety of proteins with diverse functions (transcription, DNA repair, cell cycle progression, etc.). In several instances, the FHA domain preferentially recognizes partner proteins when they are present in the phosphorylated form (5-8). Moreover, the strong specificity of the FHA domain for phosphopeptides has been clearly demonstrated by binding assays with synthetic phosphopeptides (9). Therefore, it is currently thought that the FHA domain is a general phosphopeptide recognition motif that is involved in certain phosphopeptide-mediated signal transduction pathways (10). A search for the interaction partner(s) of the FHA domain of pKi-67, which could exist in the...

The Ki-67 antigen (pKi-67) is widely used as a cell proliferation marker protein. Its actual role in the cell cycle progression, however, is presently unclear. Using a two-hybrid screening in yeast, a novel protein, termed Hklp2 (human kinesin-like protein 2), was identified and shown to interact with the forkhead-associated (FHA) domain of pKi-67. Hklp2 has 1388 amino acids and shows a striking similarity (a 53% identity in amino acids) to Xklp2, a plus-end directed kinesin-like motor found in Xenopus. The interaction domain of Hklp2 was mapped to the portion that comprised residues 1017-1237 and that was phosphorylated in vitro by incubating with mitotic but not interphasic HeLa cell extracts. That the interaction was striking in the mitotic extract was also verified. In addition, immunofluorescence using specific antibodies revealed an association between pKi-67 and Hklp2 at the periphery of mitotic chromosomes, largely in close proximity to the centromeres. These findings suggest that pKi-67 is involved in the progression of mitosis via its interaction with Hklp2.The Ki-67 antigen (pKi-67) was originally identified as an antigen for a monoclonal antibody raised against the nuclear extract from a Hodgkin's lymphoma derived cell line and is characterized as a class of proteins that are localized around mitotic chromosomes (1). Because of this, it is assumed that pKi-67 is involved in mitotic chromosome organization. Moreover, pKi-67 is a convenient cell proliferation marker, because its expression is restricted to growing cells (2). Although the recent identification and characterization of a marsupial counterpart of pKi-67, which is referred to as chmadrin, suggests a role of pKi-67 in the organization of higher order chromatin structure (3), the actual function of the molecule in the cell cycle progression is presently unclear.To better understand the role of pKi-67 in the cell cycle, a two-hybrid screening from a HeLa cDNA library was carried out using the N-terminal portion of pKi-67 as the bait. This portion is well conserved between human pKi-67 and chmadrin (the putative ortholog found in marsupial cells) and contains the FHA 1 domain. It was originally reported that the FHA domain constituted a conserved region that is found in a subset of forkhead-type transcription factors (4). The sequence profile was also found in variety of proteins with diverse functions (transcription, DNA repair, cell cycle progression, and so on) (4). In several instances, the FHA domain was shown to preferentially recognize partner proteins when they are phosphorylated (5, 6), and, thus, it is currently thought to be a general phosphopeptide recognition motif (7). A search for the interaction partner(s) of the FHA domain of pKi-67, which might be phosphorylated, is an intriguing issue because the interaction would be a significant component of the regulation of the cell cycle progression.Here, we report the interaction between the FHA domain of pKi-67 and a novel kinesin-like protein (herein referred to as Hklp2), which is a ...