We have shown that Nap1 function is regulated by phosphorylation. We observed that Nap1 interacts with several kinases, including CK2. Here, we present evidence that Nap1 is phosphorylated on several sites and is a substrate for CK2. CK2 phosphorylation appears to be necessary for efficient nuclear import of Nap1. This is the first report on the mechanism of yeast Nap1 regulation by phosphorylation. CK2 is ubiquitously expressed in the cytoplasm and nucleus, and thus, the phosphorylation of Nap1 is likely reversible, regulated by the combined action of this kinase and a phosphatase. The importance of regulated phosphorylation and dephosphorylation events is further emphasized by the fact that yeast strains expressing either the phosphorylation-defective Nap1(S159A S177A S397A) or constitutively charged Nap1(S159D S177D S397D) mutant had alterations in the cell cycle consistent with a defect in S phase progression and replication.
Functionally, members of the Nap1/SET superfamily have in common their interaction with the mitotic B-type cyclins and the ability to assemble nucleosomes onto chromatin (47
). Whether these two functions are interconnected or represent an example of a single protein performing multiple, unrelated functions is not yet understood. In higher eukaryotes, there are several members of the Nap1 superfamily to which different functions in chromatin metabolism, transcription, and cell cycle regulation have been ascribed. S. cerevisiae
has only two members of this protein family, Nap1 and Vps75, and we set out to use this simple model organism to elucidate the regulatory mechanisms governing Nap1's pleiotropic functions (52
). We identified many Nap1-interacting proteins, including most of the expected and previously characterized partners, such as histones, Kap114, and Gin4. In addition, some new partners were identified; these included the kinases CK2, Kcc4, and Cki1. Many members of the Nap1/SET superfamily in higher eukaryotes are known phosphoproteins; we show here the first evidence that yeast Nap1 is also a target for phosphorylation in vivo.
Our data show that Nap1 is a specific substrate for CK2. CK2 is a ubiquitous kinase that is highly conserved throughout eukaryotes, has a wide range of cellular targets, and is required for viability (for a review, see reference 32
). It is composed of four subunits, two catalytic α subunits and two regulatory β subunits. Interestingly, the catalytic subunits are not entirely functionally redundant. In strains carrying a temperature-sensitive allele of a single-subunit disruption of CKA1
, encoding the α subunit, prevents polarization of the actin cytoskeleton and causes a loss of cell polarity (50
). In contrast, loss of CKA2
(encoding the α′ subunit) is reported to result in elongated buds in 10 to 30% of cells and cell cycle arrests in both G1
). Both Cka2 and Cka1 were detected among our Nap1-interacting proteins. After stringent filtering, Cka1 was removed from the list but may also represent a relevant physiological interaction. The cka2
Δ strain used in this study has the CKA1
locus intact, but this strain, like the nap1
Δ strain, showed robust resistance to benomyl. Whether this phenotype is specific to the CKA2
isoform is not known. CK2 has been found in association with tubulin, centrosomes, and the mitotic spindle, offering further evidence for a role in the regulation of microtubule stability during mitosis (10
). Surprisingly, deletion of both CKA2
in our strain background led to a slight decrease in the number of cells with abnormal buds compared to the nap1
Δ strain, suggesting a synthetic genetic interaction. These results strengthen the idea that the delayed switch from polar to isotropic growth and the increased microtubule stability observed in nap1
Δ are independent phenotypes.
It has previously been shown that Drosophila
Nap1 binds to and is phosphorylated by human CK2 holoenzyme in vitro (31
). The Nap1 homolog in the nematode Steinernema feltiae
interacts with endogenous CK2 by a yeast two-hybrid (16
), and the homolog in the rice Oryza sativa
is phosphorylated by recombinant CK2 in vitro (9
). Human TSPY (testis-specific protein, Y encoded) predominantly occurs in a phosphorylated form, and a putative CK2 phosphorylation site is required for nuclear import of this protein (27
). These studies have led to speculation that Nap1 is a substrate for phosphorylation in vivo and that phosphorylation may regulate Nap1 localization. Our studies have demonstrated for the first time that this is indeed the case in S. cerevisiae
. In addition to CK2, we identified Gin4 and other kinases that associate with Nap1. Gin4 is thought to regulate bud formation through a pathway parallel to that of Nap1, since in this study and others, the deletion of both enhances the elongated bud phenotype seen in both single deletion strains (5
). Another Nim1-like kinase, Kcc4, was found to associate with Nap1, although the two proteins did not appear to interact genetically. All three proteins, Nap1, Kcc4, and Gin4, cause an elongated bud phenotype when overexpressed (4
; also data not shown). Taken together, these results suggest that these proteins function in overlapping but nonidentical pathways in the regulation of bud formation. We do not yet know whether Nap1 is a substrate for these kinases. The fourth kinase we identified in our screen for Nap1-interacting proteins was Cki1. Cki1 is involved in the synthesis of the membrane lipid phosphatidylcholine in the Kennedy pathway (20
). Cki1 and Nap1 interact genetically, as indicated by the fact that deletion of NAP1
greatly increases the benomyl sensitivity observed in the cki1
Δ mutant and exacerbates the elongated bud phenotype compared to either single deletion strain. Phosphorylation of Nap1 itself or a Nap1-interacting substrate by Cki1 may be required for normal bud formation.
Two other Nap1-interacting proteins identified were Nis1 and the newly described Nba1. Nap1 has previously been shown to bind to Nis1, whose specific function is not well understood, although it is proposed to be involved in the mitotic signaling network (24
). The nis1
Δ strain was very sensitive to benomyl relative to the wild type, implying a role for Nis1 in regulating microtubule stability. The bud neck-associated protein Nba1 is a substrate for phosphorylation by Cdc28 in vitro (57
). Global analysis of NBA1
mRNA levels during the cell cycle demonstrated that its expression, like that of Clb2, is periodic and peaks during mitosis (54
). Taken together, these data imply that Nba1 may have a cell cycle-dependent function. We show that, like Nap1, Nba1 is primarily cytoplasmic and localizes to the bud neck at G2
/M. We hypothesize that Nap1 and Nba1 interact at the bud neck and function in the regulation of G2
/M progression. As yet we have no evidence that Nba1, like Nap1, can be imported into the nucleus. However, proteomic experiments have indicated that the yeast karyopherin Kap108/Sxm1 interacts with Nba1, suggesting Kap108 may function in Nba1 nuclear import and raising the possibility that Nba1 may also shuttle between the nucleus and cytoplasm (58
Of the 11 phosphorylated residues identified on Nap1 in vivo, 3 were targets for CK2, and we have not yet determined which kinases phosphorylate the other sites. The only remaining sites that fit a consensus sequence are two in the amino terminus that are within the minimal consensus motif for phosphorylation by Cdc28. It is plausible that Nap1 is a substrate for this kinase, given the fact that Nap1 binds Clb2, the mitotic cyclin subunit for Cdc28 (25
). Future studies will focus on the functional significance of Nap1 phosphorylation at each site, and it remains possible that there are other phosphorylation sites not identified in this study.
We analyzed the potential impact of phosphorylation on Nap1 conformation using the published crystal structure (48
). Serine 82 represents the most conserved phosphorylation site identified. This site is conserved in all four human Nap1 isoforms, Nap1L1 to Nap1L4. Interestingly, comparison of the sequence surrounding this site from different species allowed us to identify a potentially novel consensus sequence. The sequence, S/T-X-Y/F-V/I, is shared from yeast to humans, including mouse, Xenopus laevis
and Schizosaccharomyces pombe
. Although we do not know as yet which kinase phosphorylates S82, this conserved sequence may constitute a novel recognition motif. S82 is in the α1 domain that is proposed to play a role in orienting the C-terminal acidic domain of the opposite subunit (48
). The acidic domain is also important for histone binding, suggesting that S82 phosphorylation may play a role in dimer stabilization or regulate the interaction of Nap1 with chromatin.
Several of the identified phosphorylated residues are located adjacent to the NES on the dimerization α2 helix (subdomain A) and the clamp domain (subdomain B). Nap1 has been predicted to be an obligate dimer; however, it is possible that phosphorylation of residues in the clamp domain leads to a change in the conformation of the clamp. As the clamp likely contributes to the formation or stability of the dimer, this may alter the interaction of the two subunits in some way (48
). In addition, two phosphorylation sites on the α2 helix appeared to be occluded by the clamp domain, suggesting that this domain must move to allow phosphorylation to occur and further supporting the notion that the clamp has more than one conformation. Park and Luger also noted three potential CK2 targets on the basis of the kinase consensus sequence (S140, S159, and S177) (48
). As S140 and S159 are located on the clamp domain, adjacent to the NES of the opposing molecule, they predicted that phosphorylation of these residues by CK2 could affect localization by increasing the accessibility of the NES. We demonstrated that S159 and S177 are phosphorylated by CK2, as well as a third residue, S397, and so far we have no evidence that S140 is phosphorylated by CK2. Serine 397 is of particular interest, since in both Oryza
Nap1 homologs, a C-terminal serine is proposed as a CK2 target (9
), and in human TSPY phosphorylation of a C-terminal serine is required for nuclear import (27
). As noted above, the location of the CK2 phosphorylation sites suggested that phosphorylation would impact export of Nap1 by altering the availability of the NES to transport factors (48
). However, we found that export was seemingly unaffected when phosphorylation was prevented, but rather nuclear import was inhibited.
The mechanism by which CK2 phosphorylation promotes nuclear import of Nap1 is not yet understood. We examined whether phosphorylation could enhance the affinity of Nap1 for its known karyopherin Kap114; however, in this study the association of recombinant Nap1 with Kap114 was unaffected by mutation of the phosphorylated residues or by in vitro phosphorylation of Nap1 (data not shown). This suggests that phosphorylation does not regulate the Kap114-Nap1 interaction. Because the Nap1 L99S mutant was not significantly redistributed to the cytoplasm in strains lacking KAP114, Nap1 likely has additional routes to the nucleus. These routes may be mediated by other karyopherins or indirectly by piggybacking onto other nuclear proteins, and it is possible that phosphorylation could promote these interactions. It is also possible that CK2 phosphorylation modulates the Nap1-histone interaction and hence regulates the formation of a stable histone-Kap114-Nap1 complex. However, we did not observe an obvious difference between the binding of the Nap1(S159A S177A S397A) or Nap1(S159D S177D S397D) to histones in an in vitro binding assay, suggesting that this was not the case.
Interestingly, we did not observe steady-state changes in the nuclear to cytoplasmic distribution of Nap1 during the cell cycle, as has been shown in higher eukaryotic homologs, although we cannot rule out the cell cycle-dependent relocalization of a small pool of Nap1 (data not shown). However, both Nap1(S159A S177A S397A) and Nap1(S159D S177D S397D) exhibited a shortened G1
and prolonged S phase relative to the wild type, with Nap1(S159D S177D S397D), which mimics constitutive phosphorylation by CK2, showing a more pronounced defect. The fact that we saw the defect with both mutants suggests that regulated cycles of phosphorylation and dephosphorylation are important for correct cell cycle progression. Loss of Nap1 is known to lead to an elongated bud phenotype and delayed G2
/M progression. In contrast, the defect we observed with the Nap1-phosphomutants was a prolonged S phase, a phenotype associated with replication stressors, such as impaired deoxynucleoside triphosphate synthesis and defects in DNA damage repair (33
). As Nap1 is a chromatin assembly and histone import factor, the observed S phase defect may be due to an alteration in the availability of histones during replication as a result of the phosphosite mutations. A cell cycle defect is also seen in yeast lacking the chromatin assembly factor, Asf1. asf1
Δ strains exhibit delays in both S phase and G2
/M and are hypersensitive to DNA double-stranded break-inducing agents, suggesting that the chromatin assembly activity of Asf1 is required during both replication and repair (56
). No role in DNA repair has yet been defined for Nap1, but it was recently shown that Nap1 greatly enhances the disassembly of nucleosomes from chromatin by RSC in vitro (35
). The RSC complex is specifically recruited to regions of double-stranded breaks, where remodeling activity makes chromatin more accessible to the repair machinery (53
). Therefore, the cell cycle defect exhibited by the Nap1 phosphomutants may in part be due to a defect in chromatin remodeling during DNA repair.
In summary, this report examines the regulation of Nap1 function, and we determine that reversible phosphorylation of Nap1 by CK2 may be involved in cell cycle regulation and the regulation of Nap1 nuclear import. The ability of Nap1(S159A S177A S397A) to rescue normal bud morphology in a nap1Δ strain implies that Nap1 phosphorylation is not required for its role in bud formation. Mechanistically, we predict that phosphorylation of Nap1 occurs in the cytoplasm prior to import, though nuclear phosphorylation is also possible, since kinase and substrate are abundant in both compartments. We propose that once Nap1 is inside the nucleus, dephosphorylation occurs, as reversible phosphorylation is necessary for timely S-phase progression, with the phosphomimic showing a greater cell cycle defect. This suggests that cycles of phosphorylation and dephosphorylation may occur in the nucleus, and it is tempting to speculate that they are necessary for Nap1 to efficiently function in chromatin assembly during S phase. Assembly factors, such as Nap1, function by binding histones and titrating them slowly onto the DNA. This requires the assembly factor to act as both histone donor and acceptor during chromatin assembly, and cycles of phosphorylation and dephosphorylation could regulate this function. In conclusion, our data show for the first time that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.