Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1(NAP1) in Drosophila melanogaster

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. Howeve r, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphopro tein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identifie d the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 ( CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser 284. Interestingly, Ser118 and Thr120 are located within a PEST domain, whi le Ser284 is adjacent to the nuclear localization signal. Substitution of t he identified phosphoresidues by alanine was found to reduce considerably t he ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to pho sphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and t he similar tryptic phospho-peptide pattern of in vitro labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2 alpha and beta subunits are as sociated with NAP1 but we found that only the catalytic alpha subunit estab lishes direct contact with NAP1 on two distinct domains of this protein. Th e location of CK2 phosphorylation sites in NAP1 suggests that their phospho rylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus. (C) 1999 Academic Press.