NAP-2: histone chaperone function and phosphorylation state through the cell cycle

We have recently cloned the human nucleosome assembly protein 2 (NAP-2). Here, we demonstrate that casein kinase 2 (CKII) from HeLa cell nuclear extracts interacts with immobilized NAP-II, and phosphorylates both NAP-2 and nucleosome assembly protein 1 (NAP-1) in vitro. Furthermore, NAP-1 and NAP-2 phosphorylation in crude HeLa cell extracts is abolished by heparin, a specific inhibitor of CKII. Addition of core histones can stimulate phosphorylation of NAP-1 and NAP-2 by CKII. NAP-2 is also a phosphoprotein in vivo. The protein is phosphorylated at the G0/G1 boundary but it is not phosphorylated in S-phase. Here, we show that NAP-2 is a histone chaperone throughout the cell cycle and that its cell-cycle distribution might be governed by its phosphorylation status. Phosphorylated NAP-2 remains in the cytoplasm in a complex with histones during the G0/G1 transition, whereas its dephosphorylation triggers its transport into the nucleus, at the G1/S-boundary, with the histone cargo, suggesting that binding to histones does not depend on phosphorylation status. Finally, indirect immunofluorescence shows that NAP-2 is present during metaphase of HeLa and COS cells, and its localization is distinct from metaphase chromosomes.