VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle

Cantarero, Lara; Sanz-García, Marta; Vinograd-Byk, Hadar; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A.

doi:10.1038/srep10543

Download PDF

Article
Open access
Published: 12 June 2015

VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle

Lara Cantarero^1,2,
Marta Sanz-García¹,
Hadar Vinograd-Byk³,
Paul Renbaum³,
Ephrat Levy-Lahad³ &
…
Pedro A. Lazo^1,2

Scientific Reports volume 5, Article number: 10543 (2015) Cite this article

3686 Accesses
31 Citations
1 Altmetric
Metrics details

Subjects

Abstract

Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome and propose a model of CB dynamics.

The coilin N-terminus mediates multivalent interactions between coilin and Nopp140 to form and maintain Cajal bodies

Article Open access 12 October 2022

Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation

Article 25 March 2021

A viral biomolecular condensate coordinates assembly of progeny particles

Article 05 April 2023

Introduction

Cajal bodies (CBs), discovered by Ramón y Cajal in 1903^1,2, are dynamic nuclear organelles without a membrane and that are enriched in several nuclear proteins and RNA-protein complexes. However, signalling pathways controlling CBs organization and function are not known. CBs play an important role in RNA processing³, particularly those associated with splicing⁴ and assembly and maturation of small nuclear RNPs⁵. CBs contain specific RNAs, such as sca-RNA affecting its biogenesis⁶ and binding to WD40-containing proteins^7,8. Coilin is also associated to telomerase RNA biogenesis⁹ and to processing of U snRNA¹⁰. Proteins within CBs interact with snRNA, snoRNA¹¹ and also with telomerase components^12,13,14, where they might also play a regulatory role not yet understood¹⁵. The interaction of coilin with RNA is also regulated by phosphorylation¹⁶. These protein and RNA complexes located within CBs are maintained by multiple weak interactions and CBs components can exchange in a very dynamic way between this organelle and the nucleoplasm¹⁷. Structurally, CBs are organized and assembled on coilin that plays a scaffold role and is the main structural constituent of CBs^18,19, but coilin is also present in smaller Histone-locus Bodies (HLB)¹⁵. Coilin is an 80 kDa (p80) protein that self-associates to form CBs²⁰, but how this aggregation is regulated in proliferating cells is not known. In addition, CBs contain multiple proteins whose function is not well known^21,22.

Cajal bodies have a dynamic structure during cell cycle, in which they assemble and reassemble²³ by a regulatory mechanism that is not known. CBs number and size are maximal at G1/ S, but are absent in mitosis and in arrested cells^18,24. Thus, CBs dynamic assembly and disassembly requires regulatory mechanisms that are not yet identified, but in which protein phosphorylation is very likely to play an important role. Coilin undergoes several posttranslational modifications and its dynamic changes are likely to be regulated by these covalent modifications, including phosphorylations^25,26 and methylations²⁷. Coilin is a hyperphosphorylated protein^{4,25,28,29,30} in cell proliferation²⁶. However, coilin protein levels remain constant throughout cell cycle progression³¹. These coilin phosphorylations are likely to regulate its interaction with other proteins²⁵ and thus contribute to different functional roles depending on the particular phosphorylated residue and the interaction partner affected that is affected. Up to now, only two unrelated kinases cdk2/cyclinE complex , which is recruited to assembled CBs³² and VRK1³³ have been shown to directly phosphorylate coilin. VRK1 is a nuclear kinase regulated in cell cycle progression³⁴ and might be a candidate to regulate CB dynamic changes in structure and composition in proliferating cells and during cell cycle progression. In cell cycle progression, VRK1 is necessary for the exit of G0 and entry in G1 and its activity and levels increase early in the G1 phase^34,35 reaching its highest level in G2/M and facilitating chromatin condensation³⁶. VRK1 is one of the most abundant nuclear Ser-Thr kinases³⁷ and hyperphosphorylates human coilin in at least eight residues, including Ser184³³. Nuclear VRK1 specifically phosphorylates and regulates histone H3^36,38, hnRNP³⁹ and several transcription factors including p53^40,41,42,43, CREB⁴⁴, c-Jun⁴⁵ and ATF2⁴⁶. VRK1 also regulates other mitotic processes⁴⁷, including Golgi fragmentation⁴⁸ and nuclear envelope assembly in mitosis^49,50. In addition VRK1 also interacts with and regulates, proteins implicated in DNA-damage responses such as 53BP1⁵¹. The kinase activity of VRK1 can also be inhibited by protein-protein interactions with some nuclear proteins as histones H2A1.2 in interphase⁵², the small GTPase RAN-GDP³⁸ and the MKP2 phosphatase⁵³. The role of VRK1 in multiple nuclear processes where nucleic-acid-protein complexes are necessary suggests that VRK1 is likely to be an important candidate to play a regulatory role in CB dynamic assembly. Moreover, depletion of coilin^54,55 or VRK1^56,57,58,59 also partially share a common phenotype of reduced fertility.

In this work we have studied the role that VRK1 plays in the regulation of CBs. We have identified that VRK1 regulates CBs assembly in cell cycle by a specific phosphorylation and also protects coilin from ubiquitin mediated degradation in the proteasome, in which the ubiquitin ligase Mdm2 is implicated. This effect of VRK1 on coilin stability regulates the assembly and disassembly of Cajal bodies in nuclei as well as its variation in cell cycle progression.

Results

VRK1 is not present in Cajal bodies but its depletion causes a loss of Cajal bodies

Cajal bodies are present in many cell types and its number ranges from two to six depending on the cell line used (Supplementary Fig. 1A). Because VRK1 is a very abundant nuclear Ser-Thr kinase³⁷ we tested whether the VRK1 protein was also present within CBs. For this aim, CBs were isolated from nuclei and the presence of coilin and VRK1 was determined by immunofluorescence and immunoblot (Fig. 1A). Purified CBs were detected by their positivity for coilin (Fig. 1A, detail and immunoblot), but VRK1 was not detected within CBs, thus if VRK1 is present its level must represent a very minor subpopulation. However, a complex between endogenous VRK1 and coilin was detected by immunoprecipitation in lysates from non-synchronized cells (Fig. 1B). This VRK1-coilin stable protein interaction was confirmed using transfected proteins in pulldown and immunoprecipitation assays (Supplementary Fig. S1B, C).

CBs form macromolecular complexes in the nucleus. Therefore, we studied the size of the protein complexes containing coilin and VRK1. For this purpose, cell lysates were prepared under mild conditions and were fractionated by HPLC. Coilin was located in two main fractions of high (700 kDa) and mid (160 kDa) molecular size (Fig.1C). VRK1 was mostly detected in the midsize fraction (Fig.1C), but a minor fraction was also detectable in the high and low MW fractions. However, VRK1 knock-down resulted in the reduction of the coilin protein present in the high molecular size fractions (6 to18) and its increase in the midsize fractions (24–36) (Fig. 1C). This midsize complex may be a basic building block for initiating CBs assembly. These results are consistent with the requirement of VRK1 for facilitating assembly or maintaining coilin within high molecular size complexes.

Coilin is a scaffold protein and it is likely that kinases phosphorylating coilin, such as VRK1³³, might contribute to the assembly of CBs. Therefore, we determined if VRK1 knock-down could affect the organization of CBs in three cell lines, HeLa (Fig. 1D), MCF7 (Supplementary Fig. S1D, Supplementary Fig. S2) and neuroblastoma SH-SY5Y cells (Supplementary Fig. S1D). In all of them depletion of VRK1 by knockdown resulted in the loss of CBs. Nuclear coilin becomes detectable in individual cells when it is concentrated by formation of CBs.

VRK1 kinase activity is necessary for assembly of Cajal bodies

Because VRK1 activity is regulated in cell cycle³⁴ and also phosphorylates coilin in multiple residues³³ we tested whether the kinase activity of VRK1 is necessary for the assembly of CBs. For this aim endogenous VRK1 was knocked down and the rescue of CBs formation was studied by expressing either wild type or kinase-dead (K179E) murine VRK1 (Fig. 2); or alternatively by wild type or kinase-dead (K179E) human VRK1 constructs that are resistant to siRNA (Supplementary Fig. S3). The active murine VRK1, but not its kinase-dead mutant (K179E), also phosphorylates coilin in Ser184 (Fig. 2B).The wild type VRK1 proteins, either murine or the siRNA resistant human protein, were able to rescue formation of CBs (Fig. 2, Supplementary Fig. S3). However, murine and human kinase-dead VRK1 (K179E) proteins were unable to recover CBs (Fig. 2, Supplementary Fig. S3). These results are consistent with the requirement of the VRK1 kinase activity for the assembly of CBs.

Coilin phosphorylation in Ser184 is associated to the kinase activity of VRK1 in proliferating cells

The requirement of the VRK1 kinase activity for CBs formation suggested that it might be implicated in CBs assembly during proliferation since both, VRK1^34,51 kinase-activity and CBs formation^4,31, are cell cycle regulated. Coilin is a hyperphosphorylated protein in proliferating cells⁴ and one of eight residues phosphorylated by VRK1 is Ser184³³. To address if VRK1 affects Ser184 in proliferating cells, MCF7 cells were placed in media with and without serum, endogenous VRK1 was immunoprecipitated and its activity determined in kinase assays that include VRK1 autophosphorylation and in the phosphorylation of histone H3 (Fig. 3A) and coilin (Fig. 3B) as substrates. Because the activity of VRK1 was lower in the absence of serum than in its presence, we determined if serum deprivation could also result in loss of CBs with respect to control (Fig.3 C–E). Both, serum deprivation and VRK1 knockdown resulted in the loss of CBs (Fig.3 C–E) and of the specific phosphorylation of coilin in Ser184, which were detected in HeLa and MCF7 cells (Fig. 3F). These data indicated that the loss of CBs in arrested cells is associated to the kinase activity of VRK1 (Fig. 3).

Coilin phosphorylation in Ser184 occurs before assembly of CB

Next we studied whether coilin phosphorylated in Ser184 was present within CBs and whether it co-localizes with VRK1 in two cell lines. Coilin phosphorylated in Ser-184 was detected within CBs, but not all CBs have Ser184-phosphorylated coilin as shown by the fluorescence profile (Fig. 4A). VRK1 was always detected in nucleoplasm and did not co-localize with phosphorylated coilin within CBs (Fig. 4B). This result indicated that after CBs assembly, this specific residue Ser184 may be dephosphorylated and was not required for CBs maintenance. Nevertheless, coilin within CBs is likely to be regulated by additional phosphorylation in multiple residues that are likely to play different functional roles.

Next we determined whether the specific phosphorylation of coilin in Ser184 was associated to cell cycle progression (Fig. 5A). For this aim, cells were synchronized with thymidine-nocodazole. The pattern of CBs formation and of coilin phosphorylated in Ser184 is inverted. Coilin Ser184 phosphorylation was mainly detected in mitotic cells, from G2/M to early G1 phase in which there are no CBs and disappeared when CBs were reassembled, consistent the requirement of this specific residue before assembly, but not for CBs maintenance (Fig. 5A). This phosphorylation of coilin in Ser184 was also studied by immunoblot analysis after thymidine-nocodazole release. Coilin-Ser184 was phosphorylated in G2/M and after its release it lasted just up to the time in which CBs were reassembled (Fig. 5B). This result is identical to the observation made in immunofluorescence experiments (Fig. 5A, bottom).

VRK1 depletion reduces coilin levels by facilitating its proteasomal degradation

To elucidate the mechanism by which VRK1 can regulate CBs disassembly we initially determined the temporal effect of VRK1 knock down on the intracellular levels of VRK1 and coilin. The reduction of VRK1 was clearly detectable at three days and preceded the reduction of coilin, which was detected two days later (Fig. 6A). Reduction of endogenous coilin protein levels was similarly achieved with three different siVRK1 in HeLa and MCF7 cells (Supplementary Fig. S5A). This late effect of VRK1 depletion on coilin levels might be either a consequence of downregulation of COIL gene expression, or of altering coilin protein stability. Taking into account that VRK1 cooperates with several transcription factors⁴⁷ we first determined whether VRK1 could regulate COIL gene expression. Depletion of VRK1 by knock down had no effect on COIL RNA expression that was determined by qRT-PCR (Supplementary Fig.S5B). To confirm this effect HeLa cells were transfected with Coilin-myc plasmid expressing coilin from a different promoter and in this case the knockdown of VRK1 also resulted in a reduction of transfected coilin protein levels (Supplementary Fig.S5C). Based on these observations we concluded that VRK1 affects the stability of coilin protein, but not COIL gene expression.

To identify the potential mechanisms by which VRK1 regulates coilin stability we thought that phosphorylated coilin might be more resistant to proteolytic degradation. To test this possibility, VRK1 was knocked down and cells were treated with proteasomal inhibitors, MG132 and lactacystin and their effect on CBs was determined. Both proteasomal inhibitors were able to prevent the disassembly of CBs as a consequence of the loss of VRK1 (Fig. 6B, Supplementary Fig.4D). This result is also consistent with the protective effect of MG132 and lactacystin on CBs in serum deprived cells (Fig. 6C), in which VRK1 is not active (Fig. 2). Therefore, we concluded that coilin is likely to be degraded in the proteasome and that this degradation is prevented by VRK1, consistent with the requirement for an active kinase.

Coilin is an ubiquitinated protein

The protective effect of proteasomal inhibitors on CBs suggested that coilin is an ubiquitinated protein. Therefore, we searched for the presence of ubiquitinated coilin. To initially address this point, MCF7 cells were transfected with plasmids expressing coilin-myc, HA-Ubiquitin and mdm2 and some cells were also treated with MG132, a proteasomal inhibitor. In the lysate, ubiquitinated proteins were detected in cells transfected with HA-Ubiquitin (Fig. 7A). Furthermore, an increase in mostly mono-ubiquitinated and some bi-ubiquitinated, coilin in the coilin immunoprecipitate were also detected in the presence of MG132 (Fig. 7A, bottom). In a reciprocal experiment ubiquitinated proteins were immunoprecipitated and coilin was also detected in this immunoprecipitate (Fig. 7B).

Cells have multiple ubiquitin ligases⁶⁰. Therefore, we tested ubiquitin ligases that likely candidates to be coilin regulators. Mdm2 and RNF8 were selected because of their implication in processes that are regulated by VRK1 such as p53⁴⁰ and DNA damage responses^51,61. For this aim, HeLa and MCF7 cells were transfected with coilin-myc and plasmids expressing either mdm2 or RNF8 ubiquitin ligases using endogenous ubiquitin. Mdm2, but not RNF8, was able to induce degradation of coilin (Fig. 7C) without affecting VRK1 (Fig.7C). Next we tested whether mdm2 or RNF8 were able to form a detectable complex with coilin. For this aim cells were transfected with tagged Mdm2 or RNF8 in combination with coilin. Mdm2 (Supplementary Fig.S6A), but not RNF8 (Supplementary Fig.S6B), was able to interact with coilin in reciprocal immunoprecipitations. To determine which ubiquitin residue is used in the initial ubiquitination of coilin, plasmids expressing ubiquitin mutated in all lysine residues, except K48 or K63 were used. Coilin ubiquitination was only detected when ubiquitin reacting only by K48 was used, but not with ubiquitin reacting by K63 (Fig. 7D, left). The use of this residue was confirmed using ubiquitin plasmids, in which all lysines were available for reaction, except the mutated K48R or K63R. In this case only the wild type or K63R mutant ubiquitinated coilin, since in both K48 is available for ubiquitination. This was confirmed by using the K48R mutant that did not ubiquitinate coilin (Fig. 7D, right). These data indicated that ubiquitin K48 is the residue used in the ubiquitination of coilin.

Blocking nuclear export prevents degradation of ubiquitinated coilin

Proteasomal degradation is a complex process that takes place either in cytosol or nuclei. To determine where coilin was being degraded, endogenous VRK1 was knocked-down and cells were treated with leptomycin B at 0.2 nM to inhibit the nuclear export of proteins. This treatment with leptomycin B resulted in the accumulation of coilin within nuclei, but not in CBs (Fig. 8A). Thus we determined whether there was an accumulation of ubiquitinated coilin within nuclei of cells that were treated with leptomycin B. For this aim cells were transfected with ubiquitin and mdm2 and nuclear export inhibited with leptomycin B, under these conditions ubiquitinated coilin accumulated in the nuclear fraction (Fig. 8B). Also the nuclear accumulation of endogenous coilin was observed in cells treated with leptomycin B (Fig. 8C). A similar accumulation of coilin within nuclei was detected in cells treated with leptomycin, but not by MG132, following VRK1 knockdown or serum deprivation (Fig. 8D). Therefore we concluded that coilin is ubiquitinated in the nucleus, but its degradation in the proteasome requires its exit to the cytosol.

Discussion

Coilin is a hyperphosphorylated protein in proliferating cells⁴. In this work we have identified the mechanisms by which VRK1 regulates the assembly and stability of CBs by phosphorylation of coilin. Coilin forms a molecular complex of 160 kDa (Fig. 1B), which is not affected by VRK1 depletion and is likely to represent the basic unit from which CBs are assembled in a VRK1-dependent manner. In this context kinases which are regulated in cell cycle are likely candidates. This process is dependent on the kinase activity of VRK1 since kinase-dead VRK1 is unable to rescue the disintegration of CBs due to VRK1 knockdown. The regulation of VRK1 activity in cell cycle and the parallel phosphorylation of coilin suggest a role for VRK1 in proliferation- dependent assembly of CBs and are consistent with a nuclear phosphorylation of coilin before CBs assembly. VRK1 is regulated in cell cycle and its expression and activity increases in the G1/S phase³⁴ and predicts that effects of serum deprivation are likely to be similar to VRK1 depletion. In this context, VRK1 specific phosphorylation of coilin in Ser184³³ is associated to cell proliferation in the presence of serum and to the regulation of VRK1 activity. However, other kinases have been identified within assembled CBs, such as CDK2/cyclin E³² and which are likely to participate in interactions and regulation of processes taking place within CBs. Since coilin can be phosphorylated in multiple residues^4,28, the pattern of coilin phosphorylation within CBs is likely to determine the role of coilin in different biological processes taking place within CBs as well as its stability. No clear role has been attributed to most of the residues phosphorylated in coilin. Phosphorylation of Ser184, one of the residues targeted by VRK1³³, has been associated to cell cycle regulation and occurs mainly in mitosis and precedes assembly of CBs (this report). Coilin Ser489 has been implicated in the interaction of coilin with RNA¹⁶ and can also contribute to CBs disruption³⁰. Phosphorylation of coilin in its C-terminal region also modulates its interactions with SMN or SmB’²⁵. VRK1 by phosphorylating coilin contributes to its stability and thus to nucleation of CBs bodies on coilin. Additionally phosphorylated coilin residues can contribute to the retention of other proteins, such as IntS4⁶² or Fam118B⁶³ whose loss also results in CBs disintegration. Furthermore, these additional phosphorylations might be an active mechanism for retaining proteins as part of CBs, such as SMN1 or gemins among others^17,22. CBs are associated with the splicing machinery and perhaps sequestration of splicing machinery in CBs during cell cycle progression might be cellular mechanisms by which transcription and translation are switched to a replication mode, with its subsequent reorganization of chromatin structure, a role that is also dependent on VRK1 activity³⁶.

Loss of coilin in cells arrested by serum deprivation is a likely consequence of its degradation. Coilin is able to interact with mdm2/hdm2 ubiquitin ligase that ubiquitinates coilin resulting in its proteasomal degradation as demonstrated by its sensitivity to inhibitors, such as MG132 and lactacystin. Since VRK1 and mdm2 do not appear to accumulate in CBs, the effect of VRK1 might be due to its effect on nuclear coilin not incorporated to CBs, or alternatively released from them. The consequence of nuclear export and degradation of coilin displaces the equilibrium of CBs to their disassembly, since CB formation is a consequence of coilin levels. VRK1 knockdown, as well as serum deprivation, facilitated coilin ubiquitination. The ubiquitin residue used in the reaction partly determines the fate of the ubiquitinated protein⁶⁰. Ubiquitination by mdm2 is mostly associated to nuclear export⁶⁴ and ubiquitination by the K48 residue is mainly associated with degradation reaction⁶⁵. Both affect coilin and these may partly account for its degradation in the cytoplasm. Blocking nuclear export with leptomycin B resulted in the retention and detection of ubiquitinated coilin dispersed within nuclei (Fig. 8). The use of leptomycin B at a concentration one-hundred and fifty fold higher resulted in coilin accumulation within nucleolus⁶⁶. There are more than six hundred ubiquitin ligases⁶⁰, indicating that ubiquitination is a selective and highly regulated process, with specificity is not yet fully understood. This separation in different compartments allows ubiquitination to have alternative functional roles depending both on number and branching of ubiquitinated residues, as well as the resulting conformational changes that can regulate its interactions with other proteins⁶⁷.

The regulation of CBs assembly in proliferating cells can have functional consequences for this organelle or its components. One of them is making accessible, or sequestering, different proteins depending on the phase of cell cycle. These proteins are likely to be free or in a different organization during interphase and be sequestered and concentrated at the time of mitosis; in that way cells can switch from a transcription to a replication mode¹⁷. Among protein candidates to be regulated by sequestration in CBs are components of splicing and RNA processing machineries, which might have a specific and different roles in cell cycle or non-dividing cells. For example, SMN is a CB component that in arrested cells is free in nuclei and cytosol, but that is partly sequestered in CBs during cell proliferation⁶⁸. Furthermore, CBs can also concentrate factors implicated in telomere maintenance¹⁷ and thus be different depending on the proliferation situation.

We propose a model, in which VRK1 mainly controls the stability of nuclear coilin, which if stabilized, can lead to its aggregation and assembly of CBs (Fig. 9). In case that VRK1 is inactive, it will permit a drop in nuclear coilin and facilitate disassembly of CB and release of its components. Thus, it will be necessary to study the fate of other CB components such as SMN1 in the absence of VRK1 since they might provide clues to its role in the pathogenesis of a complex neurological phenotype associated to VRK1 mutations and that cause a pontocerebellar hypoplasia accompanied by muscular atrophy and ataxia^69,70,71,72. VRK1 might be a common nexus among neurological phenotypes associated to the motor neuron and opens up an important research area.

Materials and Methods

Plasmids

Plasmids expressing active and kinase-dead human VRK1, pCEFL-HA-VRK1 and pCEFL-HA-VRK1(K179E) have been described previously^{38,41,42,48,51}. Plasmid pCEFL-HA-VRK1(R391/R393/V394) resistant to si-VRK1-01 has been described³³. Murine VRK1 was expressed from plasmid pCMV6-mVrk1-myc-DKK (OriGene, Rockville, MD). A kinase-dead construct of the murine VRK1(K179E) was generated with the Quick-Mutagenesis system (Stratagene, San Diego, CA) using the following primers (forward: 5′ -GTGCACGGGGACATCGAGGCCTCCAACCTGCTCCT -3′; reverse: 5′ -AGGAGCAGGTTGGAGGCCTCGATGTCCCCGTGCAC -3′. Coilin was expressed from plasmid pCMV6-coilin-myc-DKK (Origene, Rockville, MD); mdm2 from pCOC-Mdm2-X2 (M. Oren, Rehovot, Israel)⁴³; ubiquitin from pSSK-HA-Ubiquitin and pHis-Ubiquitin (D. Lane, Dundee, UK) and HA-RNF8 (T. Thomsom, IBMB-CSIC, Barcelona, Spain)⁷³. Ubiquitin was expressed from different constructs: pcDNA3- 6xHis-Ubiquitin(wt), pcDNA3-6xHis-Ubiquitin K48R, pcDNA3-6xHis-Ubiquitin K63R, (J. Lozano, University of Malaga, Spain); Ubiquitin-HA-His K63 (all Lys mutated except K63) and Ubiquitin-HA-His K48 (all Lys mutated except K48)⁷⁴.

Cell culture and transfection

The following cell lines were used in this work: SH-SY5Y, HEK293T, HeLa, MCF7, WS1, HCT116 and A549 all obtained from the ATCC-LGC (Teddington, UK). Cells were authenticated and grown as recommended by supplier.

VRK1 knock-down

VRK1 knockdown was performed using three different siRNA specific for VRK1 (Accession number NM_003384): siVRK1-01 (siV-01), siVRK1-02 (siV-02), siVRK1-03 (siV-03) and siVRK1-09 (siV-09) (all from Dharmacon RNA Technologies- GE Healthcare). The sequences targeted by these VRK1 siRNA oligonucleotides were siVRK1-01: GAAAGAGAGTCCAGAAGTA; siVRK1-02: CAAGGAACCTGGTGTTGAA; si-VRK1-03: GGAAUGGAAAGUAGGAUUA; and siVRK1-09: AGGUGUACUUGGUAGAUUA. As negative control the “ON-TARGETplus siCONTROL Non-targeting siRNA” (siCt) (Dharmacon) was used. The efficiency of RNAi transfection was determined with “siGLO RISC-free siRNA” (DHARMACON) labelled with a red fluorochrome. All of them have been previously used^34,48,51.

Briefly, cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent” (Invitrogen) according to manufacturer instructions. After transfection cells were processed for specific experiments at the times indicated in them. For rescue experiments, cells were transfected with the indicated siRNA using Lipofectamine 2000 Reagent and 36 hours later, cells were retransfected with plasmids using JetPI reagent (Poly Plus, Ilkirch, France) according to manufacturer instructions. Targeted protein and plasmid expression were analysed thirty-six hours after the second transfection⁵¹.

High-performance liquid chromatography (HPLC)

Cells were lysed 72 hours post-transfection in Lysis Buffer (20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 10% v/v glycerol, 1% Triton-X-100, protease and phosphatase inhibitors). Insoluble material was eliminated by centrifugation at 13200 rpm 20 minutes in an Eppendorf 5415R centrifuge. Soluble material was quantified and 3 mg of protein were fractionated by HPLC in a Superose 12 10/300 GL column (Pharmacia-GE Healthcare). Fractions were eluted in Elution Buffer (50 mM Tris HCl, pH 7.5, 1 mM EDTA, 100 mM KCl, sodium azide 0.025%), previously filtered (0.22 μm) and degassed, at a flow rate of 0.4 ml/min and monitored at 280 nm. Fractions of 0.2 ml were collected, precipitated and resolved on polyacrylamide gels and the following molecular weight markers (BioRad) were used: bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B12 (1.35 kDa).

Nuclei isolation

HeLa cells were plated on 100 mm dishes and 48 hours later the cells were washed and harvested by centrifugation at 600 g for 5 minutes. Pelleted cells were resuspended in 10 volumes of RSB buffer (0.01 M NaCl, 1.5 mM MgCl₂, 0.01 M Tris HCl pH 7.4) and incubated on ice 10 minutes. At this point a small aliquot was collected to examine the cells in a microscope (if the cells have “ballooned” swollen cytoplasm, the preparation is ready for the next step). Then, cells were introduced into a glass Dounce-type homogenizer and were subjected to 10-12 quick upward strokes. Once again, cells were examined in a microscope (now >90% were free nuclei). Suspension was centrifuged at 1000 g for 3 minutes (4 °C) to pellet the nuclei. The pellet was washed three times with RSB buffer.

Cajal Body Isolation

All the procedures were performed on ice. S1 solution (0.25 M sucrose, 10 mM MgCl₂) was added to HeLa nuclei to reach a final volume of 30 mL. 15 mL of Solution 2 (0.35 M sucrose, 0.5 mM MgCl₂) were added in two 50 mL Falcon tubes and resuspended nuclei were overlayed onto Solution 2 (15 mL/Falcon). These tubes were centrifuged at 1430 g for 5 minutes (4 °C) and the supernatant was decanted carefully. The pellets were resuspended in 30 mL of Solution 2 and each solution was divided in three 15 mL Falcon tubes to improve sonication (3 × 6 seconds). In this step, the solution was examined in the microscope (nuclei should be lysed and the nucleoli should be visible). The aliquots were pooled together in a 50 mL Falcon tube and 0.42x volume of 2.55 M sucrose was added (final concentration 1 M). The mixture was divided into two portions, in 50 mL Falcon tubes (approximately 20 mL each portion) and were centrifuged at 3000 g for 10 minutes (4 °C). The supernatant (contains the nucleoplasm with Cajal bodies) was collected with pipette. After that, 0.82x volume of SP1 Buffer (1 M sucrose, 34.2% Percoll, 22.2 mM Tris-HCl pH 7.4, 1.11 mM MgCl₂) and 0.05x volume of 20% Triton were added. This mixture was divided into pre-cooled SW41 ultracentrifuge tubes and they were centrifuged at 37000 rpm for 2 hours (4 °C). The pellet was transferred into a 15 mL Flacon tube and 600 units of DNase1 were added for 1 hour incubation at RT in a rotating wheel. After the incubation with DNase1, 0.05x volume of heparin (10 mg/mL) was added to the mixture. At this point, 1x volume of SP2 (20% Percoll, 10 mM Tris-HCl pH 7.4, 1% Triton X100, 0.5 mg/ml heparin) was added and incubated in a rotating wheel for 1 minute. The mixture was loaded in tubes for SW55 rotor and centrifuged at 45000 rpm for 1 hour. After centrifugation, three bands were visible in the tube and, the middle band containing the Cajal Bodies was collected. Now, 10x volume of HT Buffer (10 mM Tris-HCl pH 7.4, 1% Triton X100, 0.5 mg/ml heparin) was added and incubated in a rotating wheel. This mixture was divided in Eppendorf tubes and centrifuged at 14000 rpm for 15 minutes. The supernatants were decanted and the pellets were resuspended in 500 μl of HT Buffer. The pellets were pooled together in one tube and centrifuged at 14000 rpm for 15 minutes. Again the supernatant was eliminated and the pellet was resuspended in 500 μl of S3 solution (0.5 M sucrose, 25 mM Tris-HCl pH 9.0). This mixture was centrifuged at 8000 rpm for 5 minutes and the supernatant was centrifuged another time in the same conditions and transferred to a new tube. The supernatant was diluted with 10x volume of S3 solution and centrifuged at 14000 rpm for 15 minutes. Once more, the supernatant was eliminated and the pellet was resuspended in S3 solution and centrifuged at 14000 rpm for 15 minutes. Finally, the supernatant was eliminated; leaving 20 μl in the tube in which the pellet was resuspended obtaining the fraction enriched in Cajal Bodies.

Antibodies

The following antibodies, type and dilution were used. VRK1 polyclonals VE and VC (1:1000 for immunoblots)^{33,41,48,51,75,76}; mAb 1B5 or 1F6 (1:1000 immunoblots; 1:200 immunofluorescence)^{33,41,48,51,75,76}. VRK1 (Sigma, HPA000660, 1:200 for immunofluorescence). Flag epitope (mAb M5 at 1:1000 or polyclonal F7425 at 1:1000, both from Sigma); HA epitope (Santa Cruz: mAb sc-7392 at 1:1000 for immunoblots; Sigma H6908 at 1:200 for immunofluorescence and at 1:1000 for immunoblots). β-actin (mAb C-15 at 1:5000 for immunoblots). Histone H3 (Cell Signaling 9715 at 1:1000 for immunoblots). Mdm2 (Pierce, Ab PA5-27237 at 1:1000 for immunoblots). GARS (Santa Cruz, sc-98614, at 1:200 for immunoblots and 1:50 for immunofluorescence). Coilin (Santa Cruz, sc-56298, Pdelta; sc-32860, H300). Coilin-Ser184P was affinity purified with a Ser184P phospho-peptide (APS, Shefford, UK, at 1:1000 for immunoblots and 1:200 for immunofluorescence). RB (Santa Cruz, sc-50; 1:1000, phospho RB (Ser807/811)(Cell Signaling, 1: 1000 for immunoblots) , cyclinD1 (Santa Cruz, sc-450 at 1:500 for immunoblots). The secondary antibodies used for immunoblots using chemiluminiscence: α-mouse IgG, horseradish peroxidase-linked species-specific whole antibody (Amersham-GE Healthcare) or α-rabbit IgG (whole molecule) peroxidase conjugate (SIGMA-ALDRICH) and were developed with an ECL kit (Amersham-GE Healthcare). The secondary antibodies used for immunofluorescence in a Li-Cor Odyssey system (Thermo-Fisher) were goat α-Mouse IgG, DyLight^TM 680 and/or goat α-Rabbit Ig-G, DyLight^TM 800 (Thermo scientific).

Confocal immunofluorescence microscopy

Cells were plated on 35 mm dishes with coverslips and transfected 24 hours later with expression plasmids or siRNA. Two or three days after, respectively, the slides were collected and the cells were fixed with 3% paraformaldehyde for 30 min at room temperature. Then the cells were treated with a solution of glycine 200 mM for 15 min and then permeabilized with PBS 0,2% Triton X100 for 30 min. The cells were blocked with 1% BSA in PBS for 1 hour. Proteins of interest were detected by immunostaining with specific antibodies that were incubated overnight or for 1 hour depending on commercial instructions. The secondary antibodies were incubated together (goat anti-mouse Cy3 or Cy2 and goat anti-rabbit Cy3 or Cy2 from Jackson ImmunoResearch) for 1 hour at room temperature. Finally cells were stained with DAPI (4´, 6´-diamidino-2-phenylindole) (Sigma) 1:1000 in PBS for 10 min at room temperature, the cells were washed with PBS and slides were mounted with Mowiol (Calbiochem-Merck, Darmstadt Germany) and analysed with a LEICA SP5 DMI-6000B confocal microscope. The lasers used were: Argon (488 nm), DPSS (561 nm) and UV Diode (405 nm). Images were captured with a 63.0x lens zoomed in 1.5 − 3×with a 1024 × 1024 frame and 600 Hz scanning speed. Microscope scanner settings were maintained constant for image capture for all samples. Images were analysed with ImageJ (NIH, http://rsb.info.nih.gov/ij) and LEICA LAS AF (Leica) software.

Rescue experiments

Rescue of defective Cajal Body formation was determined using VRK1 constructs resistant to siRNA. HeLa cells were transfected with siVRK1-01 to knock down endogenous human VRK1 or with siVRK1-02 (in rescue experiments with murine VRK1), or with siControl, thirty-six hours later cells were retransfected with 3 μg of plasmid HA-VRK1(R391/R393/V394), with 3 silent substitutions that render the cDNA insensitive to si-VRK1-01⁵¹ or with murine VRK1. Rescue was also done with the same plasmids containing the K179E substitution that makes murine and human VRK1 kinase inactive⁴⁸.Thirty-six hours after retransfection cells were immunostained with anti-HA or anti-myc polyclonal antibodies to identify cells expressing exogenous VRK1. Cajal Bodies were visualized by staining with Coilin monoclonal antibody Pdelta (SantaCruz). Efficiency of endogenous VRK1 silencing and expression of siRNA-resistant VRK1 was determined by Western blot. The quantification of the number of Cajal Bodies in rescue experiments was performed using ImageJ software (NIH).

Immunoprecipitation and GST pull-down assays

Cells were plated on 100 mm dishes and harvested 48 hours after transfection. The cells were lysed with lysis buffer (1%Triton X100, 200 mM NaCl, 5 mM EDTA, 50 mM Tris pH 8.00, 1 mM NaF, 1 mM sodium orthovanadate and protease inhibitors: 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM PMSF) and after incubation on ice for 20 minutes, the extracts were centrifuged at 13200 rpm for 20 minutes at 4 °C. 40 μg of total protein lysate was analysed in SDS-polyacrylamide gel and protein expression checked by Western blot. For immunoprecipitation, 1-2 mg of total lysate was incubated with the specific antibodies for 6 to 8 hours, followed by incubation with Gamma-Bind G Plus Sepharose beads (Amersham) overnight at 4 °C^38,42. Beads were softly washed with lysis buffer 4-5 times, then resuspended in Laemmli Buffer, heated at 100 °C and analysed by SDS-Page. For GST pull-down assays, 1 mg of total cell extract was incubated with Glutathione-Sepharose beads (Amersham) for 6 hours. Beads were washed 4-5 times with lysis buffer, resuspended in Laemmli Buffer and heated at 100 °C. The proteins were analysed by SDS-PAGE.

Kinase assay

Kinase assays were performed as previously reported^33,77. Briefly, MCF-7 cells were transfected with pCMV6-Coilin-Myc-Flag or with an empty vector. 24 hours after transfection medium without FBS was added and 48 hours later the cells were harvested and proteins were immunoprecipitated. Kinase assays were performed in a final volume of 53 μl containing kinase buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 150 mM KCl), 5 μM ATP and 5μCi of [32P]ATP[γP] (PerkinElmer). In each reaction 1 mg of immunoprecipitated protein and 1 μg of recombinant Histone 3 were used ⁷⁷. The reactions were incubated at 30 °C for 30 minutes in a Thermomixer (Eppendorf). After the incubation, samples were boiled in Laemmli buffer at 100 °C for 5 minutes. The phosphorylated proteins were analyzed by electrophoresis in 10% or 12,5% SDS-polyacrylamide gels. Proteins were transferred to Immobilon-P membranes (Millipore) and the incorporated radioactivity was measured.

Immunoblots

Total protein extracts were quantified using a BIORAD Protein assay kit (Biorad, Hercules, CA). Proteins were fractionated in an SDS-polyacrylamide gel and transferred to a PVDF Immobilon-P or Immobilon-FL membranes (Millipore). The membrane was blocked with TBS-T buffer (25 mM Tris, 50 mM NaCl, 2.5 mM KCl, 0.1% Tween-20) and 5% defatted-milk. Afterwards filters were rinsed with TBS-T buffer and the specific primary antibody (indicated in individual experiments) added and incubated for 90 minutes at room temperature. The filter was rinsed and incubated with the corresponding conjugated secondary antibody. Signals were detected by chemiluminescence (ECL, Amersham) or fluorescence (Li-Cor Odyssey).

qRT-PCR

RNA was analysed and quantified using a Bioanalyzer 2100 nano-lab chip from Agilent Technologies (Germany). 100 ng of total RNA were used in a one-step reverse transcription real-time PCR amplification reaction using the Quantitec SYBR Green RT-PCR kit from Qiagen in an iCycler (BioRad, Hercules, CA). The reaction was analysed with iCycler software (BioRad). The primers used for coilin amplification were: (forward: 5′-TTACCCCCAGCAAGGGCAAGACCTC-3′; reverse: 5′-GGCCTACTGTCTTTAAGAAACCCGCAGCA-3). GAPDH amplification was used as internal control (GAPDH-F: 5′-GGTCTTACTCCTTGGAGGCCATGT-3′; GAPDH-R: 5′-ACCTAACTACATGGTTTACATGTT-3′). VRK1 was amplified with primers (F: 5′-GAGGCCATACAGACCCGTTC-3′; R: 5′-TCCACCTCGCAAGACTCACA-3′)

Drugs and treatments

Proteasome inhibitors MG132 and Lactacystine were used at 20 μM for 20 hours or at 5 μM for 10 hours, respectively. The nuclear export inhibitor Leptomycin B was used at 0.2 nM for 24 hours.

Additional Information

How to cite this article: Cantarero, L. et al. VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle. Sci. Rep. 5, 10543; doi: 10.1038/srep10543 (2015).

References

Ramon y Cajal, S. Un sencillo metodo de coloracion selectiva del reticulo protoplasmatico y sus efectos en los diversos organos nerviosos de vertebrados e invertebrados. Trab. Lab. Invest. Biol. (Madrid) 2, 129–221 (1903).
Google Scholar
Ramón y Cajal, R. El núcleo de las células piramidales del cerebro humano y de algunos mamíferos. Trab. Lab. Invest. Biol. (Madrid) 8, 27–62 (1910).
Google Scholar
Cioce, M. & Lamond, A. I. Cajal bodies: a long history of discovery. Annu. Rev. Cell. Dev. Biol. 21, 105–131, 10.1146/annurev.cellbio.20.010403.103738 (2005).
Article CAS PubMed Google Scholar
Carmo-Fonseca, M., Ferreira, J. & Lamond, A. I. Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure. J. Cell Biol. 120, 841–852, 10.1083/jcb.120.4.841 (1993).
Article CAS PubMed Google Scholar
Spector, D. L. SnapShot: Cellular bodies. Cell 127, 1071, 10.1016/j.cell.2006.11.026 (2006).
Article PubMed Google Scholar
Enwerem, II et al. Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis. Biol. Open 3, 240–249, 10.1242/bio.20147443 (2014).
Article CAS PubMed PubMed Central Google Scholar
Tycowski, K. T., Shu, M. D., Kukoyi, A. & Steitz, J. A. A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles. Mol. Cell 34, 47–57, 10.1016/j.molcel.2009.02.020 (2009).
Article CAS PubMed PubMed Central Google Scholar
Filipowicz, W. & Pogacic, V. Biogenesis of small nucleolar ribonucleoproteins. Curr. Opin. Cell Biol. 14, 319–327 (2002).
Article CAS PubMed Google Scholar
Broome, H. J. & Hebert, M. D. Coilin displays differential affinity for specific RNAs in vivo and is linked to telomerase RNA biogenesis. J. Mol. Biol. 425, 713–724, 10.1016/j.jmb.2012.12.014 (2013).
Article CAS PubMed Google Scholar
Broome, H. J. & Hebert, M. D. In vitro RNase and nucleic acid binding activities implicate coilin in U snRNA processing. PLoS ONE 7, e36300, 10.1371/journal.pone.0036300 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Lamond, A. I. & Spector, D. L. Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol 4, 605–612, 10.1038/nrm1172 (2003).
Article CAS PubMed Google Scholar
Jady, B. E., Bertrand, E. & Kiss, T. Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal. J. Cell Biol. 164, 647–652, 10.1083/jcb.200310138 (2004).
Article CAS PubMed PubMed Central Google Scholar
Cristofari, G. et al. Human telomerase RNA accumulation in Cajal bodies facilitates telomerase recruitment to telomeres and telomere elongation. Mol. Cell 27, 882–889, 10.1016/j.molcel.2007.07.020 (2007).
Article CAS PubMed Google Scholar
Venteicher, A. S. et al. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science. 323, 644–648, 10.1126/science.1165357 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Nizami, Z., Deryusheva, S. & Gall, J. G. The Cajal Body and Histone Locus Body. Cold Spring Harbor Perspect. Biol. 2, a000653 10.1101/cshperspect.a000653 (2010).
Article CAS Google Scholar
Broome, H. J., Carrero, Z. I., Douglas, H. E. & Hebert, M. D. Phosphorylation regulates coilin activity and RNA association. Biol. Open 2, 407–415, 10.1242/bio.20133863 (2013).
Article CAS PubMed PubMed Central Google Scholar
Machyna, M., Heyn, P. & Neugebauer, K. M. Cajal bodies: where form meets function. Wiley Interdiscip. Rev. RNA 4, 17–34, 10.1002/wrna.1139 (2013).
Article CAS PubMed Google Scholar
Andrade, L. E. et al. Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J. Exp. Med. 173, 1407–1419 (1991).
Article CAS PubMed Google Scholar
Raska, I. et al. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp. Cell Res. 195, 27–37, 10.1016/0014-4827(91)90496-H (1991).
Article CAS PubMed Google Scholar
Hebert, M. D. & Matera, A. G. Self-association of coilin reveals a common theme in nuclear body localization. Mol. Biol. Cell. 11, 4159–4171 (2000).
Article CAS PubMed PubMed Central Google Scholar
Saitoh, N. et al. Proteomic analysis of interchromatin granule clusters. Mol. Biol. Cell 15, 3876–3890, 10.1091/mbc.E04-03-0253 (2004).
Article CAS PubMed PubMed Central Google Scholar
Fong, K. W. et al. Whole-genome screening identifies proteins localized to distinct nuclear bodies. J. Cell. Biol. 203, 149–164, 10.1083/jcb.201303145 (2013).
Article PubMed PubMed Central Google Scholar
Dundr, M. et al. In vivo kinetics of Cajal body components. J. Cell. Biol. 164, 831–842, 10.1083/jcb.200311121 (2004).
Article CAS PubMed PubMed Central Google Scholar
Misteli, T. The concept of self-organization in cellular architecture. J. Cell. Biol. 155, 181–185, 10.1083/jcb.200108110 (2001).
Article CAS PubMed PubMed Central Google Scholar
Toyota, C. G., Davis, M. D., Cosman, A. M. & Hebert, M. D. Coilin phosphorylation mediates interaction with SMN and SmB’. Chromosoma 119, 205–215, 10.1007/s00412-009-0249-x (2010).
Article CAS PubMed Google Scholar
Hebert, M. D. Phosphorylation and the Cajal body: modification in search of function. Arch. Biochem. Biophys. 496, 69–76, 10.1016/j.abb.2010.02.012 (2010).
Article CAS PubMed PubMed Central Google Scholar
Hebert, M. D., Shpargel, K. B., Ospina, J. K., Tucker, K. E. & Matera, A. G. Coilin methylation regulates nuclear body formation. Dev. Cell 3, 329–337, 10.1016/S1534-5807(02)00222-8 (2002).
Article CAS PubMed Google Scholar
Olsen, J. V. et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3, ra3, 10.1126/scisignal.2000475 (2010).
Article CAS PubMed Google Scholar
Hearst, S. M. et al. Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines. J. Cell Sci. 122, 1872–1881, 10.1242/jcs.044040 (2009).
Article CAS PubMed PubMed Central Google Scholar
Carrero, Z. I., Velma, V., Douglas, H. E. & Hebert, M. D. Coilin phosphomutants disrupt Cajal body formation, reduce cell proliferation and produce a distinct coilin degradation product. PLoS ONE 6, e25743, 10.1371/journal.pone.0025743 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Andrade, L. E., Tan, E. M. & Chan, E. K. Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation. Proc. Natl. Acad. Sci. USA 90, 1947–1951, 10.1073/pnas.90.5.1947 (1993).
Article ADS CAS PubMed PubMed Central Google Scholar
Liu, J. et al. Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies. J. Cell Sci. 113(Pt 9), 1543–1552 (2000).
CAS PubMed Google Scholar
Sanz-Garcia, M. et al. Substrate profiling of human vaccinia-related kinases identifies coilin, a Cajal body nuclear protein, as a phosphorylation target with neurological implications. J. Proteomics 75, 548–560, 10.1016/j.jprot.2011.08.019 (2011).
Article CAS PubMed Google Scholar
Valbuena, A., Lopez-Sanchez, I. & Lazo, P. A. Human VRK1 is an early response gene and its loss causes a block in cell cycle progression. PLoS ONE 3, e1642, 10.1371/journal.pone.0001642 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Klerkx, E. P., Lazo, P. A. & Askjaer, P. Emerging biological functions of the vaccinia-related kinase (VRK) family. Histol. Histopathol. 24, 749–759 (2009).
CAS PubMed Google Scholar
Kang, T. H. et al. Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells. Mol. Cell. Biol. 27, 8533–8546, 10.1128/MCB.00018-07 (2007).
Article CAS PubMed PubMed Central Google Scholar
Varjosalo, M. et al. Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS. Nat. Methods 10, 307–314, 10.1038/nmeth.2400 (2013).
Article CAS PubMed Google Scholar
Sanz-Garcia, M., Lopez-Sanchez, I. & Lazo, P. A. Proteomics identification of nuclear Ran GTPase as an inhibitor of human VRK1 and VRK2 (vaccinia-related kinase) activities. Mol. Cell. Proteomics 7, 2199–2214, 10.1074/mcp.M700586-MCP200 (2008).
Article CAS PubMed PubMed Central Google Scholar
Choi, Y. H., Lim, J. K., Jeong, M. W. & Kim, K. T. HnRNP A1 phosphorylated by VRK1 stimulates telomerase and its binding to telomeric DNA sequence. Nucleic Acids Res. 40, 8499–8518, 10.1093/nar/gks634 (2012).
Article CAS PubMed PubMed Central Google Scholar
Lopez-Borges, S. & Lazo, P. A. The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein. Oncogene 19, 3656–3664, 10.1038/sj.onc.1203709 (2000).
Article CAS PubMed Google Scholar
Vega, F. M., Sevilla, A. & Lazo, P. A. p53 Stabilization and accumulation induced by human vaccinia-related kinase 1. Mol. Cell. Biol. 24, 10366–10380, 10.1128/MCB.24.23.10366-10380.2004 (2004).
Article CAS PubMed PubMed Central Google Scholar
Valbuena, A., Blanco, S., Vega, F. M. & Lazo, P. A. The C/H3 domain of p300 is required to protect VRK1 and VRK2 from their downregulation induced by p53. PLoS ONE 3, e2649, 10.1371/journal.pone.0002649 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Valbuena, A., Castro-Obregon, S. & Lazo, P. A. Downregulation of VRK1 by p53 in Response to DNA Damage Is Mediated by the Autophagic Pathway. PLoS ONE 6, e17320, 10.1371/journal.pone.0017320 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Kang, T. H., Park, D. Y., Kim, W. & Kim, K. T. VRK1 phosphorylates CREB and mediates CCND1 expression. J. Cell. Sci. 121, 3035–3041, 10.1242/jcs.026757 (2008).
Article CAS PubMed Google Scholar
Sevilla, A., Santos, C. R., Barcia, R., Vega, F. M. & Lazo, P. A. c-Jun phosphorylation by the human vaccinia-related kinase 1 (VRK1) and its cooperation with the N-terminal kinase of c-Jun (JNK). Oncogene 23, 8950–8958, 10.1038/sj.onc.1208015 (2004).
Article CAS PubMed Google Scholar
Sevilla, A., Santos, C. R., Vega, F. M. & Lazo, P. A. Human vaccinia-related kinase 1 (VRK1) activates the ATF2 transcriptional activity by novel phosphorylation on Thr-73 and Ser-62 and cooperates with JNK. J. Biol. Chem. 279, 27458–27465, 10.1074/jbc.M401009200 (2004).
Article CAS PubMed Google Scholar
Valbuena, A., Sanz-Garcia, M., Lopez-Sanchez, I., Vega, F. M. & Lazo, P. A. Roles of VRK1 as a new player in the control of biological processes required for cell division. Cell. Signal. 23, 1267–1272, 10.1016/j.cellsig.2011.04.002 (2011).
Article CAS PubMed Google Scholar
Lopez-Sanchez, I., Sanz-Garcia, M. & Lazo, P. A. Plk3 interacts with and specifically phosphorylates VRK1 in Ser342, a downstream target in a pathway that induces Golgi fragmentation. Mol. Cell. Biol. 29, 1189–1201, 10.1128/MCB.01341-08 (2009).
Article CAS PubMed Google Scholar
Nichols, R. J., Wiebe, M. S. & Traktman, P. The vaccinia-related kinases phosphorylate the N’ terminus of BAF, regulating its interaction with DNA and its retention in the nucleus. Mol. Biol. Cell. 17, 2451–2464, 10.1091/mbc.E05-12-1179 (2006).
Article CAS PubMed PubMed Central Google Scholar
Gorjanacz, M. et al. Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly. Embo J. 26, 132–143, 10.1038/sj.emboj.7601470 (2008).
Article CAS Google Scholar
Sanz-Garcia, M., Monsalve, D. M., Sevilla, A. & Lazo, P. A. Vaccinia-related Kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage. J. Biol. Chem. 287, 23757–23768, 10.1074/jbc.M112.353102 (2012).
Article CAS PubMed PubMed Central Google Scholar
Kim, W. et al. Macro Histone H2A1.2 (MacroH2A1) Protein Suppresses Mitotic Kinase VRK1 during Interphase. J. Biol. Chem. 287, 5278–5289, 10.1074/jbc.M111.281709 (2012).
Article CAS PubMed Google Scholar
Jeong, M. W., Kang, T. H., Kim, W., Choi, Y. H. & Kim, K. T. Mitogen-activated protein kinase phosphatase 2 regulates histone H3 phosphorylation via interaction with vaccinia-related kinase 1. Mol. Biol. Cell 24, 373–384, 10.1091/mbc.E12-06-0456 (2013).
Article CAS PubMed PubMed Central Google Scholar
Walker, M. P., Tian, L. & Matera, A. G. Reduced viability, fertility and fecundity in mice lacking the cajal body marker protein, coilin. PLoS ONE 4, e6171, 10.1371/journal.pone.0006171 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Tucker, K. E. et al. Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product. J. Cell. Biol. 154, 293–307, 10.1083/jcb.200104083 (2001).
Article CAS PubMed PubMed Central Google Scholar
Wiebe, M. S., Nichols, R. J., Molitor, T. P., Lindgren, J. K. & Traktman, P. Mice deficient in the serine/threonine protein kinase VRK1 are infertile due to a progressive loss of spermatogonia. Biol. Reprod. 82, 182–193, 10.1095/biolreprod.109.079095 (2010).
Article CAS PubMed Google Scholar
Choi, Y. H. et al. Vaccinia-related kinase 1 is required for the maintenance of undifferentiated spermatogonia in mouse male germ cells. PLoS ONE 5, e15254, 10.1371/journal.pone.0015254 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Kim, J., Choi, Y. H., Chang, S., Kim, K. T. & Je, J. H. Defective folliculogenesis in female mice lacking Vaccinia-related kinase 1. Sci. Rep. 2, 468, 10.1038/srep00468 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Schober, C. S., Aydiner, F., Booth, C. J., Seli, E. & Reinke, V. The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis. Mech. Dev. 128, 178–190, 10.1016/j.mod.2011.01.004 (2011).
Article CAS PubMed Google Scholar
Komander, D. The emerging complexity of protein ubiquitination. Biochem. Soc. Trans. 37, 937–953, 10.1042/BST0370937 (2009).
Article CAS PubMed Google Scholar
Lopez-Sanchez, I. et al. VRK1 interacts with p53 forming a basal complex that is activated by UV-induced DNA damage. FEBS Lett. 588, 692–700, 10.1016/j.febslet.2014.01.040 (2014).
Article CAS PubMed Google Scholar
Takata, H., Nishijima, H., Maeshima, K. & Shibahara, K. The integrator complex is required for integrity of Cajal bodies. J. Cell Sci. 125, 166–175, 10.1242/jcs.090837 (2012).
Article CAS PubMed Google Scholar
Li, Y. et al. Fam118B, a newly identified component of Cajal bodies, is required for Cajal body formation, snRNP biogenesis and cell viability. J. Cell Sci. 127, 2029–2039, 10.1242/jcs.143453 (2014).
Article CAS PubMed PubMed Central Google Scholar
Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229, 10.1146/annurev-biochem-060310-170328 (2012).
Article CAS PubMed Google Scholar
Jacobson, A. D. et al. The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome. J. Biol. Chem. 284, 35485–35494, 10.1074/jbc.M109.052928 (2009).
Article CAS PubMed PubMed Central Google Scholar
Carvalho, T. et al. The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body. J. Cell. Biol. 147, 715–728, 10.1083/jcb.147.4.715 (1999).
Article CAS PubMed PubMed Central Google Scholar
Ye, Y. et al. Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266–270, 10.1038/nature11722 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Hebert, M. D., Szymczyk, P. W., Shpargel, K. B. & Matera, A. G. Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein. Genes Dev. 15, 2720–2729, 10.1101/gad.908401 (2001).
Article CAS PubMed PubMed Central Google Scholar
Renbaum, P. et al. Spinal muscular atrophy with pontocerebellar hypoplasia is caused by a mutation in the VRK1 gene. Am. J. Hum. Genet. 85, 281–289, 10.1016/j.ajhg.2009.07.006 (2009).
Article CAS PubMed PubMed Central Google Scholar
Gonzaga-Jauregui, C. et al. Mutations in VRK1 associated with complex motor and sensory axonal neuropathy plus microcephaly. JAMA Neurol. 70, 1491–1498, 10.1001/jamaneurol.2013.4598 (2013).
Article PubMed PubMed Central Google Scholar
Najmabadi, H. et al. Deep sequencing reveals 50 novel genes for recessive cognitive disorders. Nature 478, 57–63, 10.1038/nature10423 (2011).
Article ADS CAS PubMed Google Scholar
Vinograd-Byk, H. et al. The Spinal Muscular Atrophy with Pontocerebellar Hypoplasia Gene VRK1 Regulates Neuronal Migration through an Amyloid-beta Precursor Protein-Dependent Mechanism. J Neurosci 35, 936–942, 10.1523/JNEUROSCI.1998-14.2015 (2015).
Article CAS PubMed PubMed Central Google Scholar
Lok, G. T. et al. Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation. Nucleic Acids Res. 40, 196–205, 10.1093/nar/gkr655 (2012).
Article CAS PubMed Google Scholar
Moreno, R., Sobotzik, J. M., Schultz, C. & Schmitz, M. L. Specification of the NF-kappaB transcriptional response by p65 phosphorylation and TNF-induced nuclear translocation of IKK epsilon. Nucleic Acids Res. 38, 6029–6044, 10.1093/nar/gkq439 (2010).
Article CAS PubMed PubMed Central Google Scholar
Valbuena, A. et al. Identification of a dominant epitope in human vaccinia-related kinase 1 (VRK1) and detection of different intracellular subpopulations. Arch. Biochem. Biophys. 465, 219–226, 10.1016/j.abb.2007.06.005 (2007).
Article CAS PubMed Google Scholar
Valbuena, A., Vega, F. M., Blanco, S. & Lazo, P. A. p53 downregulates its activating vaccinia-related kinase 1, forming a new autoregulatory loop. Mol. Cell. Biol. 26, 4782–4793, 10.1128/MCB.00069-06 (2006).
Article CAS PubMed PubMed Central Google Scholar
Vazquez-Cedeira, M., Barcia-Sanjurjo, I., Sanz-Garcia, M., Barcia, R. & Lazo, P. A. Differential Inhibitor Sensitivity between Human Kinases VRK1 and VRK2. PLoS ONE 6, e23235, 10.1371/journal.pone.0023235 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

L. C. and M. S-G. were funded by JAE-CSIC-Fondo Social Europeo fellowships. This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2010-14935), Ministerio de Economía y Competitividad (SAF2013-44810R, SAF2014-57791-REDC) and Junta de Castilla y León-Consejería de Educación (CSI002U14) to P.A.L; and grant (702/1335) from the Israel Science Foundation to P.R. and E.L.L.

Author information

Authors and Affiliations

Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Salamanca, Spain
Lara Cantarero, Marta Sanz-García & Pedro A. Lazo
Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, Spain
Lara Cantarero & Pedro A. Lazo
Medical Genetics Institute, Shaare-Zedek Medical Center, Hebrew University of Jerusalem Medical School, Jerusalem, Israel
Hadar Vinograd-Byk, Paul Renbaum & Ephrat Levy-Lahad

Authors

Lara Cantarero
View author publications
You can also search for this author in PubMed Google Scholar
Marta Sanz-García
View author publications
You can also search for this author in PubMed Google Scholar
Hadar Vinograd-Byk
View author publications
You can also search for this author in PubMed Google Scholar
Paul Renbaum
View author publications
You can also search for this author in PubMed Google Scholar
Ephrat Levy-Lahad
View author publications
You can also search for this author in PubMed Google Scholar
Pedro A. Lazo
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

L.C. participated in design, performing and analysis of experiments. M.S.-G. and H.V. contributed to design and performed some experiments. P.R. and E.L.-L. contributed to experimental design. P.A.L. contributed to the planning and experimental design of the work, coordination, interpretation of results and wrote the manuscript.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Electronic supplementary material

Supplementary Information

Rights and permissions

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

About this article

Cite this article

Cantarero, L., Sanz-García, M., Vinograd-Byk, H. et al. VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle. Sci Rep 5, 10543 (2015). https://doi.org/10.1038/srep10543

Download citation

Received: 13 January 2015
Accepted: 24 April 2015
Published: 12 June 2015
DOI: https://doi.org/10.1038/srep10543

This article is cited by

The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase
- Eva Monte-Serrano
- Patricia Morejón-García
- Pedro A. Lazo
Epigenetics & Chromatin (2023)
Olaparib and ionizing radiation trigger a cooperative DNA-damage repair response that is impaired by depletion of the VRK1 chromatin kinase
- Ignacio Campillo-Marcos
- Pedro A. Lazo
Journal of Experimental & Clinical Cancer Research (2019)
VRK1 functional insufficiency due to alterations in protein stability or kinase activity of human VRK1 pathogenic variants implicated in neuromotor syndromes
- Elena Martín-Doncel
- Ana M. Rojas
- Pedro A. Lazo
Scientific Reports (2019)
A novel mutation in VRK1 associated with distal spinal muscular atrophy
- Nannan Li
- Ling Wang
- Rong Peng
Journal of Human Genetics (2019)
What’s new in pontocerebellar hypoplasia? An update on genes and subtypes
- Tessa van Dijk
- Frank Baas
- Bwee Tien Poll-The
Orphanet Journal of Rare Diseases (2018)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.