Cloning and analysis of murine PRP4K and its homologues.
We cloned and sequenced the full-length gene trapped in the murine embryonic stem cell line CT143 (56
) (Fig. ). This gene encodes a basic protein (pI 10.26) of 1,007 aa with a predicted molecular mass of 117 kDa. This is larger than the previously described murine Prp4 homologue (Q61136; 496 aa), indicating that the original sequence was truncated at the N terminus (17
). Consistent with previous nomenclature, we have chosen to call this protein PRP4K for Prp4 kinase.
We also cloned the human homologue, which is similar to the protein KIAA0536 (AB011108) and shares partial homology to the putative human homologue of Prp4 kinase described by Gross et al. (17
). PRP4K homologues in D. melanogaster
, C. elegans
, and A. thaliana
were also identified (Fig. ), but none could be found in S. cerevisiae
Mammalian PRP4K contains a dual-specificity kinase domain that is 53% identical to that of S. pombe
Prp4p (Fig. ). There are other motifs in the basic N terminus that are conserved among metazoan PRP4Ks but absent in S. pombe
Prp4p. Among these is the sequence KKHK (KKHK box, Fig. ). This K/H-rich motif is also present in PACT, a snRNP-associated protein that interacts with both p53 and Rb (52
), and in POP101 (Srm160), a component of the splicing-associated interchromatin granules (41
). The second conserved region is an RS-like domain rich in arginine and serine (RS) (Fig. ). Lastly, two conserved sequence motifs, MI (DDMFA) and MII (DNWTDAEGYYRV), are found adjacent to the kinase domain of all Prp4 kinases including that of S. pombe
PRP4K is an essential gene in C. elegans.
Prp4 is essential for growth in S. pombe
. The ts
has both a cell cycle defect and accumulates pre-mRNA at the restrictive temperature (2
). To determine whether PRP4K is essential in metazoans, we disrupted the expression of the C. elegans
homologue (identifier F22D6.5) by RNA interference (RNAi) by using a 512-bp dsRNA fragment corresponding to the second exon of the predicted protein. This led to a highly penetrant early embryonic lethality in the F1
progeny, and no hatched larvae were observed (data not shown). Thus, PRP4K is essential in metazoans.
Subcellular localization of mammalian PRP4K.
To determine the subcellular localization of endogenous PRP4K, we generated antibodies against two N-terminal peptides conserved between the murine and human proteins (MRC1 and MRC2; Fig. ). The specificity of the affinity-purified antibodies was determined by Western blot analysis of GST fusion proteins containing each peptide epitope (Fig. ). A third antibody (H143), this one directed against sequences between the RS-like and the kinase domains of PRP4K (aa 504 to 688), was also generated.
FIG. 2. Characterization of PRP4K by Western blot analysis and indirect immunofluorescence. (A) Specificity of the anti-PRP4K antibody MRC2. Western blot analysis shows that affinity purified anti-PRP4K antibody MRC2 can detect the MRC2 epitope fused to GST (GST-MRC2) (more ...)
The predicted molecular mass of PRP4K is 117 kDa, but Western blot analysis with anti-PRP4K antibodies detects bands of ca. 152 kDa in nuclear extracts from human and mouse cells (Fig. ). Most PRP4K is extracted from nuclei by 400 mM KCl (Fig. ). Three major bands of 167, 152, and 147 kDa (Fig. , bands A, B, and C, respectively) are differentially extracted from nuclei with various salt concentrations. HeLa cell extracts containing recombinant T7 epitope-tagged PRP4K also produce these three bands when analyzed by Western blotting with anti-T7 antibodies (data not shown). These data suggest that PRP4K may be posttranslationally modified.
The subcellular distribution of endogenous PRP4K in human and mouse cells was analyzed by indirect immunofluorescence (Fig. ). PRP4K is distributed throughout the nucleus, excluding the nucleolus, but is enriched in multiple speckles. In addition, some endogenous PRP4K is associated with unfixed mitotic chromosomes (Fig. ; F9 and HT1080). In contrast, the splicing factor SC35 was not associated with mitotic chromosomes (data not shown). Detection of recombinant T7 epitope-tagged PRP4K in HT1080 cells produced a similar pattern of localization to endogenous PRP4K in both nuclei and on mitotic chromosomes (Fig. ; T7-PRP4K).
PRP4K is enriched in structures resembling splicing speckles, which colocalize with both SC35 (Fig. ) and with Sm antigens (data not shown). Upon the inhibition of transcription by actinomycin D or heat shock, SC35 concentrates into fewer, but larger, foci within the nucleus (53
). Under these conditions, PRP4K-enriched speckles continue to localize with SC35 (Fig. ).
FIG. 3. PRP4K is concentrated in SC35-containing splicing speckles. (A) Coimmunofluorescence with antibodies detecting PRP4K (H143) (FITC) and SC35 (Texas red) in human HT1080 cells alone, in the presence of actinomycin D (+Act D), or subjected to heat (more ...)
To confirm the localization of PRP4K and to determine the sequences necessary for localization to splicing speckles, cells were transfected with cDNAs encoding full-length protein (GFP-PRP4K) or part of the N terminus containing the RS-like domain (aa 188 to 780) (GFP-RS) tagged with GFP. Both proteins colocalize with SC35 (Fig. ). In contrast, constructs containing sequences between the RS-like and kinase domains (aa 512 to 1007), including motifs MI and MII, fail to localize to splicing speckles (data not shown). Thus, sequences within the RS-like domain of PRP4K alone are sufficient for colocalization with SC35-containing splicing speckles. T7 epitope-tagged PRP4K also localized to splicing speckles in interphase nuclei (data not shown).
PRP4K interacts with both chromatin and splicing factors in yeast two-hybrid analysis.
The subcellular localization of PRP4K suggests that this kinase might interact with splicing factors. To identify interacting proteins, yeast two-hybrid screens were carried out with the N terminus (aa 2 to 644) or C-terminal kinase domain (aa 637 to 1007) of human PRP4K as bait (Fig. ).
FIG. 4. Yeast two-hybrid analysis of human PRP4K. Yeast two-hybrid analysis of the N terminus or the C-terminal kinase domain of human PRP4K was carried out by using an adult human brain library. The region of PRP4K used in each screen is represented above each (more ...)
The PRP4K domain interacted strongly with the U5 snRNP-associated protein, PRP6 (40
). The interacting region of PRP6 (aa 169 to 841) contains 17 of the 19 tetratricopeptide repeats (Fig. ). A ts
allele of prp1
, the S. pombe
PRP6 homologue, accumulates pre-mRNA at the restrictive temperature and interacts genetically with prp4
). Furthermore, S. pombe
Prp1p has been shown to interact via yeast two-hybrid with Prp4p and is a target of this kinase both in vivo and in vitro (51
). Thus, the interaction between PRP6 and the kinase domain of PRP4K appears to be conserved.
In contrast, using the N terminus of PRP4K, which is not present in S. pombe
Prp4p, we identified a number of interacting proteins that play roles in pre-mRNA splicing, as well as those involved in chromatin remodeling and/or gene regulation. One N-terminal interacting protein was the human homologue of suppressor-of-white apricot
(SWAP), which regulates the alternative splicing of the CD45
and fibronectin genes (50
). SWAP can autoregulate splicing of its own transcript and is the prototype of a family of splicing proteins that share a SURP domain (65
). The minimal PRP4K-interacting region is C-terminal to the SURP domains and encompasses the RS domain of SWAP (aa 761 to 951) (Fig. ). We also isolated pinin, a desmosome-associated protein (48
). Although no functional connection between pinin and splicing has been demonstrated, pinin localizes to splicing speckles in both Xenopus
spp. and in mammals (5
). The PRP4K interacting peptide encodes the last 97 aa of pinin (aa 654 to 743), including approximately two-thirds of the DRS domain (domain rich in serines) (5
). Interestingly, PRP4K itself was also isolated in this screen, suggesting that it can dimerize through this N-terminal region (aa 206 to 644).
Another PRP4K N-terminal interacting protein, BRG1, is a mammalian homologue of the Drosophila
protein Brahma, which regulates homeotic gene expression in the fly and shares homology with the yeast chromatin remodeling factor Swi2/Snf2 (12
). BRG1 is the catalytic component of a mammalian Swi/Snf complex and has been implicated in the activation of several hormone-regulated genes (8
). The minimal interacting region of BRG1 (aa 249 to 581) is similar to that involved in interaction with HP1α (34
). Surprisingly, we also isolated two clones of N-CoR capable of interacting with the N terminus of PRP4K. The interacting region of N-CoR contains the first of two SANT domains (aa 385 to 525) (1
These data suggest that mammalian PRP4K is capable of interacting both with proteins involved in the regulation of splicing and also proteins that regulate gene expression through the modulation of chromatin structure. Recently, an N-CoR complex has been isolated that also contains BRG1 and the splicing factors SAP130 and SF3a120 (57
). Our two-hybrid data support the existence of such complexes, which may link transcriptional regulation, via changes in chromatin structure, to pre-mRNA splicing.
PRP4K interacts in vivo with N-CoR, BRG1, pinin, and PRP6.
To determine whether PRP4K interacts in vivo with the proteins isolated by yeast-two hybrid analysis, we performed coimmunoprecipitation (Co-IP) experiments from HeLa cell extracts. N-CoR, BRG1, PRP6, and pinin were each coimmunoprecipitated by anti-PRP4K antibody (Fig. ). Interestingly, when the reciprocal IPs were carried out by using anti-PRP6 or anti-BRG1 antibodies, only the fastest-migrating form (~147 kDa) of PRP4K could be coimmunoprecipitated (Fig. ). The pinin antibody did not work efficiently for IP.
FIG. 5. In vivo interactions of PRP4K. (A to C) Co-IP of PRP4K with pinin, N-CoR, BRG1, and PRP6. Complexes containing PRP6, BRG1, or PRP4K were immunoprecipitated from HeLa nuclear extracts with antibodies against each protein coupled to protein A- or protein (more ...)
The differentially migrating forms of PRP4K detected by Western blot analysis (Fig. ) suggest that it is posttranslationally modified. Phosphatase treatment of nuclear extracts reduces most PRP4K to the 147-kDa fast-migrating form (Fig. ). Thus, the slower-migrating forms of PRP4K are phosphorylated, and it is only the hypophosphorylated (147 kDa) form that is coimmunoprecipitated by BRG1 and PRP6 antibodies (Fig. ). PRP4K fails to coimmunoprecipitate with BRG1 from cells treated with the transcriptional inhibitor actinomycin D (Fig. ), suggesting that the interaction between BRG1 and PRP4K is transcription dependent.
PRP6, SWAP, and pinin are known to localize in splicing speckles (5
) and so are expected to colocalize with PRP4K in the nucleus. Indeed, GFP-tagged PRP6, GFP-SWAP, and myc-tagged pinin extensively colocalize with endogenous PRP4K, supporting the notion that these proteins can interact in vivo (Fig. ).
Immunoprecipitated PRP4K exhibits kinase activity in vitro.
To determine whether immunoprecipitated PRP4K is catalytically active, we carried out in vitro kinase assays. Immunoprecipitated PRP4K was able to phosphorylate both itself and several coimmunoprecipitated proteins (Fig. ). This kinase activity was absent if the immunoprecipitate was preincubated with MRC2 peptide, suggesting that it is due to PRP4K. A strongly labeled doublet (Fig. ) corresponds to the relative position of PRP4K, a finding consistent with the reported ability of mammalian PRP4K homologues to autophosphorylate (19
). Two other prominently labeled proteins correspond in size to BRG1 and PRP6 (Fig. , bands , respectively). To confirm that these proteins were indeed BRG1 and PRP6, we carried out kinase assays with BRG1 and PRP6 IP complexes, which also contain coimmunoprecipitated PRP4K (Fig. ). These immunoprecipitates do exhibit kinase activity, and both phosphorylated PRP6 and BRG1 are much more intense in their respective IP reactions, reflecting the relative increase in each substrate for PRP4K. Although a contaminating kinase could be coimmunoprecipitating with PRP4K, our data suggest that PRP4K is the kinase primarily responsible for this activity. Furthermore, Prp1p, the fission yeast homologue of PRP6, is a PRP4K substrate both in vitro and in vivo (51
). We suggest that PRP6 and BRG1 are substrates of PRP4K in mammalian cells.
FIG. 6. In vitro kinase activity of PRP4K in immunoprecipitates. IPs with antibodies against PRP4K (MRC2 antibody [PRP4K-IP]), BRG1 (BRG1-IP), or PRP6 (PRP6-IP) were subjected to an in vitro kinase assay and resolved by SDS-PAGE, and phosphorylated proteins were (more ...) PRP4K specifically associates with the U5 snRNP.
PRP6 is believed to play an important role in bridging the U4/U6 and U5 snRNPs within the tri-snRNP particle during spliceosomal maturation (38
). Our Co-IP data suggest that PRP4K and PRP6 interact in vivo. Therefore, we sought to determine whether PRP4K, perhaps through interaction with PRP6, was an snRNP-associated protein. PRP4K cosediments specifically with the 20S U5 snRNP particle containing PRP6 and the U5 snRNP-specific 200- and 220-kDa proteins (Fig. ). PRP4K is present in the U5 snRNP fractions as a nonstoichiometric component (Fig. ) compared to PRP6 or the 200K and 220K proteins. This suggests that only a subset of U5 snRNP particles contain PRP4K or that some PRP4K is lost under our purification conditions. This is perhaps not unexpected since many kinase-substrate interactions are transitory in nature. In contrast to PRP4K, PRP6 is also found in the fractions corresponding to the 25S tri-snRNP particle (38
) (Fig. ), which is delineated by the appearance of the U4/U6 snRNP-specific 90-, 61-, and 60-kDa proteins (Fig. ). These data demonstrate that PRP4K can be copurified with total snRNPs from HeLa cells and that it specifically associates with the U5 snRNP. Consistent with these results, PRP4K was also found to specifically coimmunoprecipitate the U5 snRNA from HeLa nuclear extracts (data not shown). Previously, a snRNP-associated kinase activity, called the U1 snRNP 70K protein kinase, was identified that predominantly associates with the U1 snRNP (60
). It is significant to note that PRP4K does not cosediment with the U1 snRNP (Fig. ), suggesting that it is a novel snRNP-associated kinase that is distinct from the U1 snRNP 70K protein kinase.
FIG. 7. PRP4K specifically cosediments with the 20S U5-snRNP during glycerol gradient centrifugation. (A) Proteins from each fraction of the glycerol gradient were separated by SDS-10% PAGE and visualized by Coomassie blue staining (Protein). Fractions that cosediment (more ...) PRP4K is a component of the N-CoR-2 complex.
It has recently been demonstrated that N-CoR exists as part of two multiprotein complexes which exhibit deacetylase activity and contain distinct subunits (57
). One of the complexes contains both BRG1 and the splicing factors SAP130 and SF3a120 (57
). Based on our identification of N-CoR, BRG1, and several splicing factors as proteins interacting with PRP4K by yeast two-hybrid analysis, it was of interest to determine whether PRP4K is a constituent of the biochemically purified N-CoR-1 and N-CoR-2 complexes (Fig. ). Western blot analysis indicated that PRP4K coeluted from a gel filtration column with N-CoR, and after immunoaffinity chromatography, it was found to be a constituent of the N-CoR-2 complex. In contrast, BRG1 and PRP6 were found in both the N-CoR-1 and the N-CoR-2 complexes. These results suggest that endogenous PRP4K can be isolated as a component of an N-CoR complex containing N-CoR, BRG1, and PRP6.
FIG. 8. Detection of the presence of PRP4K, BRG1, and PRP6 in the N-CoR-1 and -2 complexes. (A) Schematic representation of the purification of the N-CoR-1 and N-CoR-2 complexes (57). (B) Western blot analysis of the N-CoR-1 and N-CoR-2 complexes after elution (more ...) The affinity-purified PRP4K/N-CoR complex exhibits HDAC activity.
The presence of PRP4K in the N-CoR-2 complex suggested that PRP4K/N-CoR complexes exist in vivo. Such complexes might bridge or coordinate the regulation of transcription, via chromatin modification, and pre-mRNA splicing. To determine whether N-CoR, PRP4K, BRG1, and PRP6 are components of the same complex, we optimized the purification strategy to specifically isolate PRP4K/N-CoR-containing complexes from HeLa nuclei (Fig. ). After two rounds of conventional column chromatography, fractions containing both N-CoR and PRP4K were run on an S300 Sepharose gel filtration column to both size the complex and determine whether N-CoR and PRP4K would coelute as single or multiple complexes (Fig. ). The majority of N-CoR eluted from the sizing column in complexes ranging from 1.0 to 1.5 MDa, and several of these fractions also contained BRG1, PRP4K, and PRP6 (Fig. , fractions 20 to 22). Thus, it appears that large-molecular-weight complexes exist in vivo that contain N-CoR, BRG1, PRP4K, and PRP6. To further purify the complex, fractions 20 to 22 from the S300 column were pooled and subjected to immunoaffinity purification for PRP4K complexes by using an anti-PRP4K antibody column. PRP4K complexes were then acid eluted from the immunoaffinity column and subjected to Western blot analysis (Fig. ). N-CoR, PRP4K, BRG1, and PRP6 were coeluted from the immunoaffinity column, confirming the association of these proteins within the PRP4K/N-CoR complex.
FIG. 9. Affinity purification of the PRP4K/N-CoR deacetylase complex. (A) Purification scheme for the isolation of complexes enriched in both N-CoR and PRP4K. At each stage, N-CoR- and PRP4K-containing fractions were determined by Western blot analysis and pooled (more ...)
A critical function of N-CoR as a corepressor is the recruitment of HDAC activity. If PRP4K is truly a component of a functional N-CoR complex, one would expect that such complexes would exhibit HDAC activity. To determine whether the affinity-purified PRP4K/N-CoR complex was associated with HDAC activity, affinity-purified PRP4K/N-CoR complex or mock-affinity-purified complex was subjected to a liquid deacetylase assay with 3H-labeled histones prepared from HeLa nuclei (Fig. ). The affinity-purified PRP4K/N-CoR complex exhibited strong HDAC activity compared to the mock-affinity-purified complex, suggesting the PRP4K/N-CoR complex is indeed a deacetylase complex.