Regulations of the Histone Chaperone Molecules Nap1p and Nucleoplasmin by Phosphorylation

Kennedy Calvert, Meredith Emily, Department of Microbiology, University of Virginia
Pemberton, Lucy, Department of Microbiology, University of Virginia

The dynamic interaction of histones with DNA is regulated by histone chaperones, providing a mechanism for chromatin-dependent transcriptional control. I examined two disparate histone chaperones, mouse NPM2 and yeast Nap1p and determined that both are phosphoproteins and substrates for phosphorylation by CK2. The histone chaperone Nucleoplasmin is a maternally expressed protein in Xenopus laevis oocytes and is involved in histone transport and sperm chromatin decondensation in early embryogenesis. In a proteomic analysis of mouse oocyte maturation I identified the mouse homolog of nucleoplasmin (NPM2) and cloned and characterized the gene encoding NPM2. I determined that expression of NPM2 mRNA and protein is oocyte specific, and decreases rapidly following fertilization. NPM2 undergoes a change in localization during oocyte maturation that correlates with a reversible shift in molecular weight, suggestive of phosphorylation. In S. cerevisiae Nap1p has multiple functions, being a cofactor for the nuclear import of H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. I identified Nap1-interacting factors including a novel budneck associated protein, Nba1p, and showed that Nap1p and Nba1p are phosphoproteins in vivo and are phosphorylated by the mitotic kinase Cdc28p/Clb2p. I determined that the interaction of Nap1p with some of its binding partners is cell-cycle dependent, suggesting that its different functions may be regulated by the protein complexes with which it is associated. Nap1p, like NPM2, is phosphorylated by CK2 and I determined that this phosphorylation promotes its import into the nucleus. Mutation of CK2 phosphoserines renders Nap1p able to rescue normal bud formation, but unable to restore normal cell iii cycling in a Clb2-dependent strain, resulting in prolonged S phase. A constitutively charged mutant is unable to assemble chromatin, implying that reversible phosphorylation of Nap1p is required for release of histones onto chromatin. Thus, phosphorylation of Nap1p by CK2 may regulate both its localization and cellular functions. These studies show that localization of both Nap1p and NPM2 may be dependent upon phosphorylation, and these signaling events are likely to be involved in cell cycle progression. Phosphorylation of histone chaperones by CK2 may therefore represent a conserved mechanism for chromatin-dependent regulation of gene expression.

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PHD (Doctor of Philosophy)
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