Phosphorylation by casein kinase 2 regulates Nap1 localization and function

Abstract

In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.

FIG. 1.

Nap1-interacting proteins. PrA- or TAP-tagged proteins as indicated were isolated from whole-cell lysates using IgG-Sepharose and eluted with MgCl₂. The coprecipitation of Nap1 with these proteins was analyzed by Western blotting with an anti-Nap1 antibody (αNap1). For Cka2-TAP, the blot was probed with rabbit IgG antibody (αIgG) to detect Cka2-TAP followed by Nap1 antibody. The white arrowhead indicates Nap1 band.

FIG. 2.

Nap1 interacts directly with the bud neck-associated protein, Nba1. (A) MBP-Nba1 (200 nM), MBP (200 nM), GST (1 μM), GST-Nap1 (1 μM), and GST-Nap1 fragments (indicated by amino acid numbers above the lanes; 1 μM) were immobilized and incubated with 250 nM GST-Nap1 (top blot) or 500 nM MBP-Nba1 (bottom blot). Bound proteins were visualized by Western blotting with anti-Nap1 (α-Nap1) or anti-MBP (α-MBP) (for Nba1) antibodies. (B) Nba1-GFP₂ fusion protein was expressed in wild-type (WT) or nap1Δ cells and visualized by fluorescence microscopy. The coincident differential interference contrast (DIC) image is also shown. (C) Nba1 fragments (indicated by amino acid number) were expressed as GFP₂ fusion proteins in wild-type yeast and visualized as described above for panel B.

FIG. 3.

Genetic interactions of NAP1. (A) Strains of the indicated genotype were equalized, spotted at 10-fold serial dilutions, and grown on YPD plates with and without benomyl. (B) Strains of the indicated genotypes were examined by differential interference contrast (DIC) microscopy, and the percentage of budded cells exhibiting elongated buds was quantified. The error bars indicate the standard error of the means for three independent experiments. Statistically significant differences between nap1Δ and the double deletion strains are indicated as follows: *, P < 0.05; **, P < 0.01. DIC images of selected strains as indicated are shown above the bar graph. WT. wild type.

FIG. 4.

Nap1 is phosphorylated. (A) Schematic of Nap1. Arrows illustrate the relative positions of the 11 identified phosphosites, with the amino acids of the CK2 sites indicated numerically. Black bars beneath the schematic show the regions contained within the solved structure (48). (B) Tertiary structure of the Nap1 dimer (left). The subunits are shown in purple and yellow, and phosphorylated residues on both subunits are green. The labeled subdomains A and B refer to the purple subunit. Close-up view (right) of the clamp region indicates phosphorylated residues in this domain, and the likely position of S177 is shown. An alternate projection of the dimer, illustrating S82, is shown for clarity (bottom view) (48). (C) Recombinant GST-Nap1 and the indicated Nap1 mutants were incubated with purified CK2 and [γ-³²P]ATP for the time indicated. The proteins were separated by SDS-PAGE, Nap1 was visualized by Coomassie blue staining (CBB), and phosphorylation was measured by ³²P incorporation detected using a PhosphorImager. (D) In vitro kinase assay of GST-Nap1 or GST-Nap1(S159S177AS397A) (labeled Nap1 3S-A) was carried out for 30 min as described in Materials and Methods, and proteins included in the reaction mixture are as indicated. (E) In vitro kinase assay of GST-Nap1 or GST-Nap1 3S-A was carried out as described in Materials and Methods for the times indicated.

FIG. 5.

Reversible phosphorylation of Nap1 by CK2 may regulate normal progression through S phase. (A) Nap1, Nap1(S159A S177A S397A) (labeled Nap1 3S-A), or Nap1(S159D S177D S397D) (labeled Nap1 3S-D) were expressed in the Clb2-dependent strain and examined by differential interference contrast (DIC) microscopy. (B) The same strains were fixed, and the DNA was stained with Sytox green. Cells were analyzed for DNA content using the Amnis ImageStream instrument. Random images of cells within the peak corresponding to 4N DNA content (vector control sample only) are displayed on the right. BF, bright-field image, SYTOX, Sytox green-stained DNA. (C) Strains as in panel A were stained with Hoechst and observed by fluorescence and DIC microscopy and scored morphologically for their cell cycle distribution. The numbers of cells with no bud (G₁), cells with a small bud and one nucleus (S phase), and cells with a large bud and two nuclei (G₂/M) are indicated. Comparison of strains showed statistically significant differences (*, P < 0.05; **, P < 0.01). (D) The numbers in the boxes indicate the percentage of cells in each phase of the cell cycle derived from panel C.

FIG. 6.

Phosphorylation of Nap1 by CK2 does not prevent histone binding by Nap1. GST, GST-Nap1, GST-Nap1(S159A S177A S397A) (labeled Nap1 3S-A), or GST-Nap1(S159D S177D S397D) (labeled Nap1 3S-D) (all at 1 μM) was immobilized and incubated with purified core histones (1 μM). Bound proteins were visualized by Coomassie blue staining. The black asterisk indicates a band corresponding to bovine serum albumin. Inputs of each protein (25 μmol) are shown.

FIG. 7.

Phosphorylation of Nap1 by CK2 regulates nuclear import. (A) Nap1, Nap1(S159A S177A S397A) (labeled Nap1 3S-A), or Nap1(S159D S177D S397D) (labeled Nap1 3S-D) were expressed as GFP₂ fusion in nap1Δ cells and visualized by fluorescence microscopy. The coincident differential interference contrast (DIC) and Hoechst-stained images are shown. (B) Export-deficient Nap1-L99S, Nap1 3S-A-L99S, and Nap1 3S-D-L99S GFP₂ fusion proteins were expressed and visualized as described above for panel A. (C) MBP-Kap114 (500 nM) was immobilized and incubated with recombinant Nap1 or Nap1 3S-D (both 250 nM). Bound proteins were visualized by Western blotting and quantitated using the Odyssey infrared imaging system as described in Materials and Methods. Relative binding of Nap1 to Kap114 is expressed in arbitrary units (AU). (D) The indicated GFP fusions were expressed in kap114Δ cells and visualized as described above for panel A.