VRKs represent a distinct branch of the casein kinase family of serine/threonine kinases; they were so named because of their high homology to the vaccinia B1 serine/threonine kinase. The homology between these viral and eukaryotic kinases led to the hypothesis that they might have overlapping substrate specificities and hence overlapping functions. Indeed, human, mouse and Drosophila
VRK1 can complement the DNA replication defect that characterizes nonpermissive infections performed with a vaccinia virus mutant encoding a temperature-sensitive B1 protein (Boyle and Traktman, 2004
and K. Boyle and P. Trakman, unpublished results). Although there are no data implicating the VRKs in cellular DNA replication per se, siRNA depletion of C. elegans
VRK leads to an early embryonic lethality characterized by impaired nuclear division (www.wormbase.org
). Likewise, mutations in the D. melanogaster
homolog of VRK (NHK1) result in defects in chromosomal segregation during mitosis and meiosis (Cullen et al., 2005
; Ivanovska et al., 2005
). The work described herein, therefore, was stimulated by our hypothesis that the identification of substrates that could be readily phosphorylated by both VRK1 and B1 would provide important clues as to the biological roles performed by these kinases.
Phosphorylation of Whole Cell Lysates by the VRK1 and B1 Kinases
As a first attempt to identify unknown cellular substrates for VRK1 and B1, we performed in vitro kinase assays using extracts of BSC40 cells as a source of substrates. The extracts were heat-inactivated before being assayed so that only the exogenously added kinases would direct the incorporation of 32P-ATP into target substrates (). Few if any phosphorylated species were observed when no exogenous kinase was added; purified B1 kinase, in contrast, phosphorylated many proteins within these extracts. When purified VRK1 kinase was utilized in the same assay, only two phosphorylated proteins were observed. A 50-kDa species corresponding to autophosphorylated VRK1 was seen, as was a radiolabeled band migrating at ~10 kDa. Interestingly, B1 also appeared to target a protein(s) of the same electrophoretic mobility (arrow).
Figure 1. BAF is a substrate for phosphorylation by recombinant B1 and VRK1 kinases. (A) Phosphorylation of heat-killed cell lysates by B1 and VRK1 kinases. Heat-killed lysates of BSC40 cells were incubated with [γ-32P]ATP in the absence (no kinase) or (more ...)
Data generated in a yeast two-hybrid screen of the entire Drosophila
proteome had previously identified three proteins as able to interact with the Drosophila
VRK protein (Alvarez et al., 1995
; Giot et al., 2003
). One was a protein of unknown function, predicted to contain a bipartite nuclear localization sequence and to have an unusually high (17%) proline content. A second protein identified was norpA, which has been previously shown to have phospholipase activity (Alvarez et al., 1995
). The third protein, which showed the strongest interaction with dVRK, was the D. melanogaster
ortholog of the DNA-binding protein Barrier to Autointegration Factor (BAF). Mammalian BAF was originally identified as a 10-kDa cellular protein that interacted with retrovirus preinteraction complexes and prevented intramolecular recombination by the proviral DNA, thereby promoting integration of the proviral DNA into the cellular genome (Lee and Craigie, 1994
). Because BAF has a molecular weight similar to the protein we observed being phosphorylated by VRK1 and B1 in whole cell lysates (), we hypothesized that the latter protein might indeed be BAF. We therefore obtained an α-BAF antibody from K. Furukawa, which we used to perform immunoblot analyses of the same lysates shown in . Immunoreactive species were visualized by chemiluminescent detection, and phosphorylated species were visualized by autoradiographic analysis of the same filter. We observed three immunoreactive bands in the samples that had been phosphorylated by the exogenous kinases: one band comigrated with the band seen in untreated samples and the other two comigrated with the 32
VRK1 Phosphorylates Purified BAF In Vitro
The data described above strongly suggest that BAF present within cellular extracts becomes phosphorylated when incubated with the VRK1 or B1 kinases. To confirm this directly, we purified recombinant BAF in a dimeric form and used it as a substrate for in vitro kinase assays. These assays contained 40 nM VRK1, 1 μM BAF, and [γ-32
P]ATP and were performed for 0-60 min. For comparison, we performed parallel assays using a GST-p53 fusion protein as a substrate. Previous studies have described VRK1-mediated phosphorylation of GST fusion proteins containing fragments of the tumor suppressor protein p53 (Lopez-Borges and Lazo, 2000
; Barcia et al., 2002
; Vega et al., 2004
); in particular, VRK1 has been shown to phosphorylate p53 on thr1
8 (Lopez-Borges and Lazo, 2000
). The reaction products were electrophoretically resolved, silver-stained to ensure that equal levels of protein were present, and then excised from the gel and subjected to Çerenkov counting. As shown in , VRK1-mediated phosphorylation of BAF was rapid and robust. In contrast, the autoradiographic signal for GST-p53 was nearly undetectable (inset), and at 60 min, ~180-fold more radiolabel had been incorporated into BAF than into p53. Thus, VRK1 phosphorylates BAF with an efficiency that greatly exceeds what is seen for GST-p53.
BAF Is Phosphorylated on ser4 and Less Efficiently on thr2 and/or 3
The extreme N′ terminus of BAF is highly conserved in different species and contains two thr
and one ser
residues that represent potential targets for phosphorylation (thr
2,3 and ser
4). As an initial test of whether this region of the molecule might contain the site(s) of phosphorylation, we used a coupled in vitro transcription/translation system to express WT BAF and a variant in which residues 2-4 had been replaced by an ala
residue, and determined that VRK1 could phosphorylate the former but not the latter protein (our unpublished data). To precisely map the sites of N′-terminal phosphorylation and determine the biochemical impact of this modification, we generated alleles of BAF that contained the following amino acid substitutions: MTTSQ (WT) → MTTA4
Q, and MA2A3A4
Q. Using previously established protocols (Lee and Craigie, 1998
; Umland et al., 2000
), we induced the expression of these proteins in E. coli
and prepared purified preparations of the dimeric form of the WT and mutant BAF proteins. Each of the proteins analyzed displayed comparable chromatographic profiles.
VRK1-mediated Phosphorylation of BAF. We analyzed the ability of VRK1 to phosphorylate the WT and mutant forms of BAF by performing in vitro kinase assays for 0-120 min. Reactions containing 3 nM VRK1 and 3 μM BAF were resolved by SDS-PAGE and the BAF protein was visualized by silver-staining (top panels) and autoradiography (bottom panels), as shown in . For WT BAF, as the time course of phosphorylation progressed, we observed the original BAF protein (gray arrowhead) being converted to two more slowly migrating species (black and white arrowheads). The middle species (black arrowhead) was observed after only 1 min of phosphorylation, and 50% of the BAF protein was present in this form after 2 min of phosphorylation. By 10 min, all of the protein had been shifted to this middle species (corresponding to 1 phosphorylation event/~0.5 s). At this time, the most slowly migrating form (white arrowhead) had also begun to appear; at 120 min, perhaps 20% of the total protein was present in this uppermost form. The autoradiograph shows that both the middle and upper forms did indeed represent phosphorylated species. A more quantitative profile of phosphorylation is shown in , which shows a graphic representation of the level of phosphorylation determined by phosphorimage quantitation of each lane. The plot for VRK1-mediated phosphorylation of WT BAF is shown with black circles: a biphasic profile is seen. The first phase, which is of ~10 min in duration, represents rapid and robust phosphorylation, which corresponds to the generation of the middle species shown in . A second slower phase then ensues, with a gradual rise in 32P incorporation that continues until 120 min; this phase corresponds to the conversion of some of the BAF protein to the uppermost form seen in .
Figure 2. In vitro phosphorylation of recombinant BAF by human VRK1, VRK2, and vvB1. Preparations of WT (MTTSQ) and mutant (MAAAQ, MAASQ, MTTAQ, MTTDQ) BAF dimers (3 μM) were used as substrates in in vitro kinase reactions performed with hVRK1 (A and C), (more ...)
We then performed parallel analyses of the mutant forms of BAF in which the N′-terminal ser and/or thr residues had been changed to ala. When thr2, thr3, and ser4 were simultaneously changed to ala, the ability of VRK1 to phosphorylate BAF was lost (, and lines with filled squares in ). These data confirm that all of the sites phosphorylated by VRK1 lie within this extreme N′ terminus of BAF. Next, we examined the MAASQ allele, in which the two thr residues had been altered. As shown in , the middle species (black arrowhead) appeared with the same kinetics seen with WT BAF, and all of the BAF protein was converted to this species. As shown in (compare lines with filled triangles and filled circles), the initial, rapid phase of phosphorylation of the MAASQ protein was indistinguishable from what we observed for WT BAF. However, in this case, there was no second, slow phase of phosphorylation—the levels of 32P incorporated had reached a plateau after 10 min of phosphorylation, and no upper species (white arrowhead) was seen by silver staining or autoradiography. Thus, we conclude that the initial, rapid phase of phosphorylation of WT BAF (and the appearance of the middle form seen in ) represents phosphorylation of ser4; the second phase of phosphorylation represents slow and incomplete phosphorylation of thr2 and/or thr3. This conclusion was supported by the analysis of the MTTAQ protein, in which the target ser residue had been altered. Here, the rate of phosphorylation was significantly slower (6-fold reduction in slope; , open circles) and the ultimate level of 32P incorporation was approximately one-half of that seen with WT BAF. Examination of the silver-stained gel and the autoradiograph confirms that a single shifted species accumulated slowly. These data confirm that ser4 is the preferred site of phosphorylation and that phosphorylation of thr2 and/or thr3 does occur, albeit at a significantly slower rate. Finally, we examined the phosphorylation rate of the MTTDQ form of BAF, in which ser4 had been replaced with an asp in an attempt to observe the rate of thr2 and/or thr3 phosphorylation when a negative charge mimicking “prephosphorylation” was present at residue 4. Under these circumstances, the rate of phosphorylation of thr2 and thr3 is slowed even further, suggesting that the initial rapid phosphorylation of ser4 by VRK1 may interfere with subsequent phosphorylation of thr2 and/or thr3. These data may explain why only ~20% of WT BAF undergoes the thr phosphorylation events that generate the uppermost species, whereas all of the MTTAQ protein can be shifted to a phosphorylated form.
B1-mediated Phosphorylation of BAF. In light of our observation that the B1 kinase could also phosphorylate BAF in whole cell lysates, we sought to compare its target sites with those found for VRK1. Purified B1 was therefore incubated with WT and mutants forms of BAF in kinase assays similar to those described for VRK1. Again, phosphorylation was monitored both by visualizing electrophoretic shifts () and by quantitating 32P incorporation (). As we had seen with VRK1, B1 induced the formation of two phosphorylated forms of BAF that migrated more slowly during SDS-PAGE. The rate of B1-mediated phosphorylation of WT BAF was also similar: 3 nM kinase could phosphorylate 3 μM WT BAF in ~10 min, as measured by a complete shift of the unphosphorylated BAF to the middle protein species (black arrowhead; , lane 5). B1 could not phosphorylate the BAF-MAAAQ protein, indicating that the site(s) for B1-mediated phosphorylation were also contained within the extreme N′ terminus of BAF (, filled squares). Phosphorylation of the MAASQ variant by B1 closely approximated what had been seen with VRK1; the initial rate of phosphorylation was the same as for WT BAF (, filled triangles vs. filled circles), but the second, slow phase of phosphorylation was absent and hence a lower plateau was reached. Again, phosphorylation of the MTTAQ variant proceeded significantly more slowly, with a 10-fold decrease in the initial reaction rate. These results for B1 closely mirrored those found for VRK1 and indicate that B1 targets ser4 preferentially, but is able to modify thr2 and/or 3 given additional time. Finally, we assayed the rate at which B1 phosphorylates the BAF-MTTDQ variant of BAF, in order to determine whether substituting a negatively charged residue at the ser4 position would impair the phosphorylation on thr2 and/or thr3, as we had observed for VRK1. However, two shifted forms were observed after phosphorylation (black and white arrowhead, ), and the rate of phosphorylation was indistinguishable from that observed for the MTTAQ variant (, compare open triangles with open circles). Thus, the presence of the asp residue at position 4 adversely affected the phosphorylation of thr2 and/or thr3 by VRK1, but not by B1.
BAF Is Also Phosphorylated by VRK2.
We previously described the relationship of the VRK paralogs to each other and to the B1 kinase (Nichols and Traktman, 2004
). The VRK1 and VRK2 enzymes share a significant amount of identity within their catalytic domains, and both are indeed catalytically active. Although VRK1 is predominantly nuclear, VRK2 is found associated with the ER and the nuclear envelope. Because the catalytic domains of VRK1 and VRK2 are closely related, we also assayed purified VRK2 kinase for its ability to phosphorylate WT and variant preparations of BAF in vitro. These data are shown graphically in . VRK2 does indeed phosphorylate WT BAF with similar kinetics to VRK1, and moreover, the pattern of phosphorylation obtained with the various BAF mutants is indistinguishable from what was observed with VRK1 (compare ).
Phosphoamino Acid Analysis. Further confirmation that ser4 is the preferred site of phosphorylation by VRK1 and B1 was provided by performing phosphoamino acid analysis of phosphorylated WT BAF or BAF-MTTAQ. In these reactions, the total concentration of ATP was reduced eightfold to allow us to increase the specific activity and hence augment the radioactive signal. Under these more limiting conditions, only a single electrophoretically shifted species was observed after VRK1-mediated phosphorylation of WT BAF (unpublished data). The phosphoamino acid analysis indicates that VRK1 only phosphorylates ser under these conditions (, lanes 1-3). Phosphoserine could be detected by 1 min after the initiation of the kinase assay and increased in abundance throughout the 60-min time course; in contrast, even after 60 min, <1% of the radiolabel was found at the position of phosphothreonine. When the same analysis was performed on BAF-MTTAQ (, lanes 4-6), phosphoserine was no longer observed, as would have been expected from the results shown in . In contrast, the appearance of phosphothreonine was accelerated. Thus, phosphorylation of ser4 appears to diminish the efficiency with which thr 2 and/or thr3 are phosphorylated.
Figure 3. Phosphoamino acid analysis of B1- and VRK1-phosphorylated BAF proteins. WT BAF and the MTTAQ variant were phosphorylated in vitro by VRK1 or vvB1 for 1, 5, and 60 min. Reaction products were resolved electrophoretically, transferred to PVDF, excised, (more ...)
When WT BAF was incubated with B1 (, lanes 7-9), phosphoserine was detected in significant levels after 1 min of incubation and increased in intensity during the rest of the time course; phosphothreonine was observed in significant levels after 5 min of incubation and also increased substantially during the rest of the time course. As expected, we observed no phosphoserine when kinase reactions containing BAF-MTTAQ and B1 were performed. Instead, phosphothreonine was observed in significant levels after 1 min of incubation (, lane 10). Thus, the B1 kinase also phosphorylates thr2 and/or thr3 more rapidly in the absence of ser4 phosphorylation, but its overall preference for ser4 versus thr2/3 is less absolute than is VRK1s under these reaction conditions.
Phosphorylation of BAF Has a Modest Effect on Its Interaction with the LEM Domain
BAF has been shown to form specific interactions with three components of the inner nuclear envelope, Lap2, emerin, and MAN1, through a common motif known as the LEM domain (Acharya et al., 1996
; Lee et al., 2001
; Shumaker et al., 2001
; Holaska et al., 2003
; Mansharamani and Wilson, 2005
). We were interested in determining whether phosphorylation of BAF might affect its interaction with the LEM domain. Glutathione sepharose beads containing immobilized GST or GST-LEM were incubated with BAF or phosphorylated BAF, and the efficiency with which the BAF species bound to the matrix was determined by immunoblot assay (). We consistently observed that 2-3-fold more untreated BAF bound to the immobilized GST-LEM than did phosphorylated BAF. Thus, phosphorylation of BAF has a modest but reproducible impact on the BAF-LEM interaction.
Phosphorylation of ser4 and/or thr2/3 Disrupts BAF's Ability to Bind DNA
A second and important property of BAF is its ability to bind dsDNA tightly and without sequence preference. Previous structure/function studies have implicated lys
6 as participating in DNA binding (Harris and Engelman, 2000
; Umland et al., 2000
; Segura-Totten et al., 2002
); the proximity of this residue to the MTTSQ motif was provocative. We therefore probed the ability of untreated and phosphorylated BAF to bind to a DNA cellulose affinity resin at near physiological concentrations of NaCl (100 mM). BAF, 3 μM, was preincubated with or without ATP, in the presence or absence of VRK1, for 7.5 or 90 min. These time points, selected based on the time courses shown in , were chosen to represent either partially (primarily ser
4) or fully (ser
4 and thr
2/3) phosphorylated BAF. Parallel experiments were performed using the B1 kinase, except that only a 60-min time point, representing full phosphorylation of BAF, was used. Each of these samples was then incubated with DNA-cellulose, and the load and bound fractions were subjected to immunoblot analysis (). We observed that ~50% of the untreated BAF bound to the resin under these reaction conditions; prior incubation with ATP alone or kinase alone did not alter this profile. However, if BAF had been phosphorylated by either B1 or VRK1, its ability to bind to DNA was severely inhibited; <0.5% of the input BAF bound to the resin (, top panels). This effect was seen after 7.5 or 90 min of incubation with VRK1, suggesting that phosphorylation of ser
4 was sufficient to abrogate DNA binding.
To gain further insight into the relationship between phosphorylation and the loss of DNA binding, similar studies were performed with the BAF variants described above in . Untreated BAF-MTTAQ and BAF-MAASQ bound to the DNA cellulose with similar efficiency to WT BAF, and both proteins lost this ability after phosphorylation. These data indicate that phosphorylation of either ser4 or thr2/3 by VRK1 or B1 is capable of inhibiting BAF's ability to bind DNA. The DNA binding ability of BAF-MAAAQ, in contrast, was unaffected by incubation with kinase + ATP. Somewhat surprisingly, untreated BAF-MTTDQ protein bound to DNA cellulose with the same efficiency as WT BAF, despite the presence of an asp residue at the position of ser4. Apparently, the presence of a negative charge at this position does not have the same effect as acquisition of a phosphate group. However, when the MTTDQ protein was phosphorylated at thr2/3 with VRK1 or B1, it did lose its ability to bind to the DNA cellulose.
Because of the unexpected observation that BAF-MTTDQ was still able to bind to DNA cellulose under our experimental conditions, we performed additional binding studies in which WT and mutant BAF proteins were applied to the DNA cellulose resin in the presence of increasing concentrations of salt (). For WT BAF, raising the concentration of NaCl to 700 mM salt only led to a 20% decrement in the amount of protein that bound to the DNA cellulose resin. These data agree with the previous observation that BAF remains bound to retroviral PICs until >750 mM NaCl is applied (Suzuki and Craigie, 2002
). The binding of the BAF-MTTAQ protein was somewhat more salt-sensitive; increasing the salt concentration to 300, 500, or 700 mM NaCl led to 38, 73, and 81% decreases in the amount of protein that bound to the resin, respectively. The BAF-MTTDQ protein was significantly more salt-sensitive; in the presence of 300 mM salt, we observed an 80% decrease in the amount of BAF bound to the DNA cellulose, and there was a >97% reduction when the salt was raised to 500 or 700 mM NaCl. These data confirm that ser
4 has an important role in mediating the BAF-DNA interaction, and that modifications to this residue have an inhibitory impact that follows the hierarchy ser
The Phosphorylation Status and Subcellular Distribution of BAF Changes upon Overexpression of VRK1
The data described above provide strong evidence that BAF is an excellent substrate for VRK1-mediated phosphorylation and that this phosphorylation has a modest effect on the interaction of BAF with LEM-containing proteins and a dramatic effect on its interaction with DNA. To obtain preliminary corroboration that VRK1 would affect BAF in a similar manner in vivo, we generated reagents that allowed the expression of epitope-tagged VRK1 and/or BAF in cultured cells. We prepared vectors that directed the expression of 3XFLAG-BAF, 3XFLAG-BAF-MAAAQ, GFP-BAF, and GFP-BAF-MAAAQ. We also generated constructs expressing 3XFLAG-VRK1 and 3XFLAG-VRK1D177A; the amino acid substitution in the latter allele affects a critical residue involved in coordinating ATP via a Mg2+ salt bridge and hence ablates catalytic activity.
Analysis of endogenous VRK1, or transiently expressed variants containing N′ or C′ epitope tags, has revealed that the protein is found to be localized throughout the nucleus. (Lopez-Borges and Lazo, 2000
; Sevilla et al., 2004a
; Nichols and Traktman, 2004
). This nuclear localization was seen once again when we transiently expressed 3XFLAG-VRK1 on its own (unpublished data). Similar, transiently expressed GFP-BAF or GFP-BAF MAAAQ localized to the nucleus when expressed alone (), as expected from previous studies of BAF localization (Segura-Totten et al., 2002
; Shimi et al., 2004
Figure 6. Expression of WT VRK1 results in the relocalization of WT GFP-BAF to the cytoplasm of U2OS cells. U2OS cells were transfected with plasmids directing the synthesis of GFP-BAF or GFP-BAF-MAAAQ, with or without plasmids directing the synthesis of 3XFLAG-hVRK1 (more ...)
When 3XFLAG-VRK1 and GFP-BAF were introduced together, there was an increased pool of cytoplasmic BAF; indeed BAF appeared to be found throughout the cell (arrows, ). Surprisingly, in these cells, we also observed that VRK1 was now dispersed throughout the cell. This change in BAF localization was not seen when GFP-BAF was coexpressed with the catalytically null VRK1D77A, indicating that VRK1-mediated phosphorylation events were driving the relocalization of BAF. Support for the conclusion that it was the phosphorylation of BAF itself that was causing its relocalization came from our observation that the GFP-BAF MAAAQ protein retained its nuclear localization when coexpressed with active VRK1 (or the catalytically inactive variant; unpublished data). Because BAF-MAAAQ cannot be phosphorylated by VRK1, these cumulative data imply that the VRK1-mediated phosphorylation of BAF alters its cellular localization.
We next sought to gain more direct evidence that the intracellular localization of BAF was correlated with, or regulated by, its phosphorylation status. To this end, we used constructs encoding 3XFLAG BAF and first confirmed that this epitope-tagged version retained the DNA-binding properties of untagged BAF. 3XFLAG-BAF was synthesized in vitro using a coupled transcription/translation protocol and then incubated with DNA cellulose. shows that 35S-labeled 3XFLAG BAF does indeed bind to DNA cellulose. We then transiently transfected cells with the plasmid directing the expression of 3XFLAG-BAF, and metabolically labeled the cells with 32PPi in order to monitor the phosphorylation of BAF in vivo. Cell lysates were subjected to immunoprecipitation with FLAG-agarose resin, and the bound proteins were resolved electrophoretically, transferred to nitrocellulose, and visualized with both immunoblot analysis and autoradiography (). Two electrophoretically distinct forms of BAF were seen on the immunoblot (black and white arrowheads), and a single radiolabeled species that comigrated with the more slowly migrating species (black arrowhead) was observed on the autoradiograph. These data confirmed that transiently expressed BAF was indeed being phosphorylated in vivo by endogenous kinases. To examine whether this phosphorylation was affecting thr2,3 and/or ser4, we performed parallel transfections with plasmids expressing WT BAF (MTTSQ) or variants in which thr2,3 and/or ser4 were mutated to ala (MTTAQ, MAASQ, MAAAQ). The epitope-tagged proteins were retrieved by immunoprecipitation, left untreated or treated with λ phosphatase, and then resolved electrophoretically and visualized by immunoblot analysis. As shown in , the WT protein appeared as two distinct species of nearly equivalent abundance; upon phosphatase treatment, the abundance of the more slowly migrating species was dramatically reduced (compare lanes 1 and 5). A similar profile was seen for the MAASQ protein (, lanes 2 and 6), although in this case the upper band was somewhat less abundant in the untreated sample and absent after phosphatase treatment. For the MTTAQ protein, a faint but distinct upper band was observed, and this species was also eliminated by phosphatase treatment (, lanes 3 and 7). These data provided confirmation that BAF was indeed being phosphorylated in vivo, and supported the conclusion that thr 2,3 and ser4 are minor and major determinants, respectively, of the efficiency with which this modification occurs. For the MAAAQ treatment, only one species was observed, and its mobility was not affected by phosphatase treatment (, lanes 4 and 8). This variant, which was not a substrate for VRK-mediated phosphorylation in vitro (), also does not appear to undergo detectable phosphorylation in vivo.
These data also confirmed that we could use immunoblot analysis to monitor the electrophoretically distinct unphosphorylated and phosphorylated BAF proteins. We therefore used this assay to monitor the subcellular fractionation of WT BAF and the MAAAQ variant. Cells were therefore transfected with plasmids directing the expression of 3XFLAG-BAF or 3XFLAG-BAF MAAAQ, alone or with plasmids directing the expression of VRK1 or VRK1D177A
, and then subjected to a limited Triton X-100 extraction followed by centrifugation. The supernatant fraction has been shown to contain soluble cytoplasmic and nucleoplasmic proteins, whereas the pellet contains proteins associated with the nuclear matrix, intermediate filaments, and chromatin (Okuno et al., 2001
; Zaidi et al., 2001
; Dreuillet et al., 2002
; Yoshizawa-Sugata et al., 2005
). These fractions were then subjected to immunoblot analysis with α-FLAG serum (). When 3XFLAG-BAF was expressed alone, it was found in both the soluble and pellet fractions. However, whereas the soluble fraction contained equivalent amounts of the unphosphorylated and phosphorylated forms of BAF, the pellet fraction was highly enriched for unphosphorylated BAF (lanes 1 and 2). The profile was quite different when VRK1 was coexpressed with 3XFLAG-BAF. First, the soluble fraction was now significantly enriched in phosphorylated BAF (compare lanes 3 and 1), and there was a sharp decrease in the total amount of BAF present in the pellet fraction (compare lanes 4 and 2). These data indicate that, in the presence of VRK1, significantly more BAF is phosphorylated and that this protein is no longer fixed within an insoluble nuclear compartment, but is in fact soluble. As would be expected from this conclusion, expression of VRK1D177A
did not alter either the phosphorylation status or fractionation profile of BAF (compare lanes 5,6 with 1,2). When we examined the profile of 3XFLAG-BAF MAAAQ, we found a single electrophoretic species corresponding to unphosphorylated BAF; this protein, too, partitioned to both the soluble and pellet fractions. Because this form of BAF cannot be phosphorylated by VRK1, it was not surprising that we observed no impact on its electrophoretic mobility or subcellular fractionation when either VRK1 or VRK1D177A
was coexpressed (lanes 7-12). Together, these data indicate that the extreme N′ terminus of BAF undergoes phosphorylation in vivo and that this phosphorylation can be readily modulated by the VRK1 kinase. They further indicate that both soluble and insoluble pools of BAF are present within cells. It is predominantly the unphosphorylated BAF that is present in the insoluble pool, which probably corresponds to an association with the nuclear matrix/chromatin. The phosphorylated BAF is not found associated with these nuclear compartments, which is consistent with our findings that, in vitro, phosphorylation of BAF diminishes its interaction with LEM-containing proteins and virtually abolishes its interaction with DNA ().