that the phosphorylated form of Ser 2 CTD mediates the physical interaction between unphosphorylated Npl3 and RNAP II. Cka1 phosphorylates Npl3 in vitro In yeast, the cytosolic kinase Sky1 phosphorylates the SP motif closest to the C-terminus of Npl3 (S411) [10]. Phosphorylation of Npl3 at S411 in a sky1 deletion strain is reduced, but not abolished [8] (data not shown), raising the possibility that another Npl3- specific kinase remained to be identified, and that other phosphorylation sites in Npl3 might exist. To ascertain the later possibility, endogenous Npl3 was immunoprecipitated from whole cell extracts and phosphorylation sites were analyzed by mass spectrometry. Multiple phosphorylation sites were identified in the endogenous Npl3 (Table 1). Our results confirmed phosphory- lation of serine 224, 349 and 356, which where identified previously in two large-scale phosphorylation analyses [34,35]. One new additional site was shown to be phosphorylated, serine 212. This data is summarized in Table 1. We speculated that a putative kinase might be able to modulate the activity of Npl3 in the nucleus. As noted earlier, CK2 kinase had been shown to phosphorylate several targets within the cleavage/polyadenylation machinery (Cft1, Fip1, Brr5 and Pta1) [24,36]. Therefore, this kinase was a good candidate and was tested for its ability to phosphorylate Npl3 in vitro. Recombinant His-Cka1 and His-Npl3 were purified from E. coli and incubated in the presence of [c-32P] labeled ATP. Cka1 phosphorylates Npl3 and this modification is reversed by addition of Lambda- Phosphatase (Figure 3A). A combination of mass spectrometry and point mutants was used to further analyze phosphorylated peptides in Npl3. In vitro, CK2 phosphorylation resulted in modification of multiples sites in Npl3, including Ser 212, Ser 349 and Ser 356 sites identified in vivo (Table 1). Phosphorylation of S411 was tested using the S411 substitutions (S411A, S411D and S411E) in the kinase assay (Figure 3B). A significant decrease was observed in the phosphorylation of Npl3-S411A, - S411D and -S411E as compared to wild-type. The residual phosphorylation signal observed for the mutant Npl3s is probably due to phosphorylation of the other sites reported in Table 1. We conclude from these results that Npl3 is hyperphosphorylated in vivo and that S411 is likely a major target of CK2 phosphorylation. Given that the phosphorylation of sites in Npl3 did not strictly fit the known consensus sequence for mammalian CK2 ([ST]XX[EDpTpS]) [20], and that the RGG/RS1-3 peptides were not efficiently phosphorylated when tested in the in vitro CK2 Table 1. Npl3 phosphorylation sites. Npl3 (414 aa) Endogenous CK2 in vitro phosphorylated Npl3 Stimulates Elongation kinase assay (data not shown), the specificity of the yeast Cka1 was assayed by mass spectrometry using peptide libraries (Figure S1). Quantification by mass spectrometry of the phosphorylation efficiency of Cka1 confirmed the specificity of yeast Cka1 used in our study for peptides with serine or threonine residues within acidic motifs, as previously reported for the mammalian CK2 counterpart [20,37]. Conversely, peptides containing SP motifs were phosphorylated very poorly (Figure S1). In the context of the entire Npl3 protein, the SP or SR motifs of Npl3 that were detected as phosphorylated in vitro might reside in an acidic environment that favors phosphorylation by Cka1. This possibility is supported by evidence from the structure of the central RRM domain of Npl3, which revealed that all acidic residues lie on the surface of the protein [33]. Npl3’s reported self-association may also be a factor, especially given recent evidence that dimerization is in some instances required for the phosphorylation of targets with poor similarity to recognition motifs [38,39]. CK2 phosphorylates Npl3 in vivo The ability of CK2 to phosphorylate Npl3 was next tested in the context of the cellular environment using strains that have the genes for both of the catalytic subunits of CK2 deleted and carry complementing plasmids for either a wild-type (CKA1) or a mutant allele of CKA1 (cka1-12(ts) or cka1-45(ts)) [40]. These strains were grown and assayed for the steady state phosphorylation of Npl3 by immunoblotting with general anti-Npl3 and anti-phospho-S411, which was demonstrated previously to be specific for the S411 phosphorylation in vivo [10]. The wild-type CKA1 strain showed robust phosphorylation of Npl3 at both permissive (25uC) and non- permissive temperatures (37uC), whereas cka1-12 showed reduced phosphorylation at both temperatures, and cka1-45 showed a decrease upon the shift to the non-permissive temperature (Figure 3C). At steady-state-levels, Npl3 localizes to the nucleus [41]. Therefore, the phosphorylation decrease observed in the cka1 mutant strains was assumed to be due to CK2 mutations. The residual phosphorylation of S411 is presumed to be due to other kinases, including Sky1. While indirect phosphorylation of Npl3 cannot be absolutely excluded, we conclude from these results that Cka1 is required for the phosphorylation of Npl3-S411 in vivo. Phosphorylation of Npl3 affects its competition for RNA binding As mentioned earlier, phosphorylation of Npl3 by Sky1 reduces RNA binding affinity [8]. Therefore, Npl3 phosphorylation might Phosphorylated Serines Treatment Ser212, Ser224 Ser349, Ser356 Ser411 Ser77, Ser79, Ser212, Ser230, Ser328, Ser336, Ser349, Ser356 Ser411 WCE (whole cell extract) WCE WCE 32P-in vivo labeling Kinase assay Analysis MS MS-IMAC MS-IMAC 2D gel MS ‘‘ ‘‘ ‘‘ Point mutants Reference This study This study, [34] (Ficarro et al, 2002) (Gilbert et al, 2001) This study Phosphorylation sites identified in Npl3. *Highlighted residues where phosphorylation sites detected in both the endogenous and in vitro CK2 phosphorylated Npl3. doi:10.1371/journal.pone.0003273.t001 PLoS ONE | www.plosone.org 4 September 2008 | Volume 3 | Issue 9 | e3273