A novel nucleolar protein, NIFK, interacts with the forkhead associated domain of Ki-67 antigen in mitosis

In a previous study, we demonstrated that the fork-head associated (FHA) do main of pKi-67 interacts with the novel kinesin-like protein, Hklp2 (Sueish i, 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 pr otein of 293 residues as another binding partner of the FHA domain of pKi-6 7 (referred to as NIFK for nucleolar protein interacting with the FHA domai n 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 arrest ed cells. In addition, a moiety of hNIFK was co-localized with pKi-67 at th e peripheral region of mitotic chromosomes. The hNIFK domain that interacts with Ki-FHA was mapped in the yeast two-hybrid system to a portion encompa ssed by residues 226-269, In a binding assay utilizing Xenopus egg extracts , it was found that the mitosis-specific environment and two threonine resi dues within this portion of hNIFK (Thr-234 and Thr238) were crucial for the efficient interaction of hNIFK and Ki-FHA, suggesting that hNIFK interacts with KiFHA in a mitosis-specific and phosphorylation-dependent manner. The se findings provide a new clue to our understanding of the cellular functio n of pKi-67.