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The subcellular localization of -related -2 (VRK2) isoforms determines their different effect on p53 stability in tumour cell lines Sandra Blanco, Lucia Klimcakova, Francisco M. Vega and Pedro A. Lazo

Instituto de Biologı´a Molecular y Celular del Ca´ncer, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Universidad de Salamanca, Spain

Keywords VRK is a new kinase family of unknown function. Endogenous human p53; phosphorylation; Ser-Thr kinase; VRK2 vacinia-related kinase 2 (VRK2) protein is present in both the nucleus and the cytosol, which is a consequence of alternative splicing of two VRK2 Correspondence messages coding for proteins of 508 and 397 amino acids, respectively. P. A. Lazo, IBMCC-Centro de Investigacio´ n del Ca´ncer, CSIC-Universidad de Salamanca, VRK2A has a C-terminal hydrophobic region that anchors the protein to Campus Miguel de Unamuno, E-37007 membranes in the endoplasmic reticulum (ER) and mitochondria, and it Salamanca, Spain colocalizes with calreticulin, calnexin and mitotracker; whereas VRK2B is Fax: +34 923 294 795 detected in both the cytoplasm and the nucleus. VRK2A is expressed in all Tel: +34 923 294 804 cell types, whereas VRK2B is expressed in cell lines in which VRK1 is E-mail: [email protected] cytoplasmic. Both VRK2 isoforms have an identical catalytic N-terminal domain and phosphorylate p53 in vitro uniquely in Thr18. Phosphorylation Database Sequence VRK2B has been submitted to of the p53 protein in response to cellular stresses results in its stabilization the GenBank database under the accession by modulating its binding to other proteins. However, p53 phosphorylation number AJ512204. also occurs in the absence of stress. Only overexpression of the nuclear VRK2B isoform induces p53 stabilization by post-translational modifica- (Received 30 January 2006, revised 29 tion, largely due to Thr18 phosphorylation. VRK2B may play a role in March 2006, accepted 31 March 2006) controlling the binding specificity of the N-terminal transactivation domain of p53. Indeed, the p53 phosphorylated by VRK2B shows a reduction in doi:10.1111/j.1742-4658.2006.05256.x ubiquitination by Mdm2 and an increase in acetylation by p300. Endo- genous p53 is also phosphorylated in Thr18 by VRK2B, promoting its stabilization and transcriptional activation in A549 cells. The relative phos- phorylation of Thr18 by VRK2B is similar in magnitude to that induced by taxol, which might use a different signalling pathway. In this context, VRK2B kinase might functionally replace nuclear VRK1. Therefore, these might be components of a new signalling pathway that is likely to play a role in normal cell proliferation.

In the human kinome the vaccinia-related kinase is 2D213 [2] and it is CG6386 in Drosophila [3]. (VRK) protein family has been identified as a distinct The unique family homologues in Schizosaccharomyces subfamily that diverged early in evolution from the pombe and Saccharomyces cerevisiae are the Hhp1 and branch leading to the casein kinase I (CKI) group [1]. Hrr25 , respectively [4], and both implicated in This branch has 13 different proteins grouped into the response to genotoxic damage [5,6]. three subfamilies, VRK, TTBK and CKI [1]. Neverthe- The catalytic domain of VRK proteins shares less, in lower eukaryotes there is only one homologue homology with the vaccinia virus early gene, B1R [7], gene; thus in Caenorhabditis elegans, the homologue which is required for viral DNA replication [8–10]. In

Abbreviations ER, endoplasmic reticulum; VRK2, vaccinia-related kinase 2.

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2487 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al. mammals, the VRK family has three members. The critical to allow for both normal cell division and kinase domain of the VRK proteins shows relatively tumour suppression, while retaining the capacity for weak conservation [1], but is catalytically active for rapid induction in response to genotoxic stress [31,32]. VRK1 [11–14] and VRK2, although not for VRK3 Thus, its levels are tightly regulated, mainly by phos- [13]. VRK1 has a nuclear localization signal and is phorylation [33]. detected in the nucleus in some cell lines and in trans- Several kinases are implicated in the stability of p53 fected cells [11,13], but it is also present in the cytosol by targeting at least seven different residues in its in other cell lines [15], particularly in some types of N-terminal transactivation domain [34,35], each adenocarcinoma (unpublished results); however, the modulated by different types of stimulation [36–41]. regulation and coordination of the subcellular localiza- Therefore, there are functional differences regarding tion of different VRK proteins are unknown. The p53 stability or transcriptional activity depending on VRK1 protein is able to phosphorylate several tran- the residue phosphorylated [42,43]. Phosphorylation of scription factors such as p53 [11,15], c-Jun16, and human p53 at Ser15 or Ser20 (equivalent to murine ATF2 [17]. VRK1 and VRK2 share 61% identity in Ser18 and Ser23) promotes p300 recruitment and their catalytic domains, with no conservation in other therefore its acetylation by p300 [43–45], but p53 can parts of the protein, suggesting functional differences also be stabilized in the absence of phosphorylation of between the two kinases that have yet to be character- these two residues [46]. Moreover, phosphorylation of ized. It has been postulated that this protein family murine Ser18 (human Ser15) is not necessary for p53 might play a role in proliferation because they are tumour suppression [47]. Cells also need to have a highly expressed in early haematopoietic development basic mechanism that maintains a basal level of p53 in murine embryos [18], in tissues containing prolifera- in a state of readiness and able to respond to any tive cells and in tumour cells [7]. VRK1 is expressed at stress that may arise in normal life, when most cells very high levels in retinal neurons and its expression are in interphase [15]. More recently, phosphorylation decreases dramatically the first day after birth [19], it of p53 in Thr18 (Thr21 in murine p53) has acquired also correlates with proliferation markers in head and more relevance [48], because it is implicated in both neck squamous carcinomas [20]. In chronic myelogen- the p53–Mdm2 interaction and p300 recruitment. This ous leukaemia, expression of VRK1 can differentiate residue is phosphorylated by casein kinase I delta only those who will respond to imatinib treatment from when it has previously been phosphorylated at Ser15 those who will not [21]. In B cells, analysis by quanti- [49,50], but this kinase is cytosolic in interphase [51]; tative MS indicates that it is downregulated when Myc Thr18 is uniquely phosphorylated by the nuclear expression is induced [22]. VRK1 is also regulated in VRK1 protein [11,15]. Phosphorylation at Thr18 redu- response to peroxixome proliferators in murine hepato- ces binding to the p53-negative regulator Mdm2, and cytes [23]. VRK1 expression is activated in E2F and promotes its interaction with the cofactor p300. inhibited by p16 and in nonphosphorylated retinoblas- Mdm2 catalyses the ubiquitination of p53 and its tomas [24]. The data available on VRK2 are much subsequent proteolytic degradation [52–54]. p300 more limited. VRK2 is downregulated in human acetylates p53 at its C-terminus, promoting its mononuclear B cells during the innate immune transcriptional activation [55–58]. The functional con- response to bacteria [25], and upregulated in T cells by sequences of Thr18 phosphorylation are p53 stabil- CD3 ligation and if costimulated by CD28 [26]. ization and the activation of p53-dependent gene VRK1 contributes to p53 stability via two mecha- transcription [15]. Phosphorylation of Thr18 has been nisms, one of which is dependent on Thr18 phosphory- detected in cells treated with taxol and some other lation. It also appears to be implicated in the control drugs [33,59], as well as in cellular senescence [60], of normal proliferation in the absence of cellular stress, suggesting that several signalling pathways are and its inactivation by specific RNA interference involved. blocks cell division [15], consistent with observations We identified that VRK2 has different subcellular in C. elegans, where it is embryonic lethal. In adult localizations corresponding to expression of two iso- C. elegans there is a slowdown in growth suggesting forms by the human VRK2 gene. Their expression var- problems in cell-cycle progression [2]. The p53 protein ies depending on the cell type. Both isoforms have plays a major role in controlling the cell response to similar properties regarding their phosphorylation sub- many types of cellular stress [27,28], and its intracellu- strates and the specific phosphorylation of p53 in vitro. lar levels appear to determine the susceptibility of a The nuclear VRK2B isoform might be functionally cell to tumour development [29,30]. Regulation of p53 redundant with VRK1, and seems to be expressed in protein levels and transcriptional activity are therefore cells in which VRK1 is localized in the cytoplasm. In

2488 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms cells lacking nuclear VRK1 this nuclear isoform might endogenous VRK2 protein could exist in two forms, regulate p53 mainly by phosphorylation. membrane bound, as detected in the ER and mito- chondria, or free as detected in both cytosol and nuclei. Results

Localization of the endogenous human The VRK2 gene generates by alternative splicing VRK2 protein two different isoforms that differ in their C-terminus First we determined the subcellular localization of the endogenous VRK2 protein. For this, we used a specific The detection of two subcellular locations for the rabbit polyclonal antibody against full-length VRK2 known VRK2 protein suggested that these might be protein. We analysed the localization of VRK2 in two due to differences in the expression of the unique cell lines, WSI, derived from normal skin fibroblasts, human VRK2 gene. To test this possibility, VRK2 and MCF7 cells from a breast carcinoma. In both cell cDNA from HeLa cells was cloned by RT-PCR. Two lines there was strong staining in both the cytosol and cDNA sequences of 1833 and 1877 nucleotides were nucleus, and there was a particulate aspect, suggesting isolated. Comparison of these sequences with the that VRK2 might be associated with some organelle human VRK2 gene shows that they were generated by (Fig. 1A,B). To further refine the subcellular distribu- alternative splicing. Isoform B had an additional exon tion, three additional markers were included; the endo- of 44 nucleotides, designated new exon 13 that changed plasmic reticulum (ER) was identified by detecting the reading frame and included an early termination calnexin (Fig. 1A), mitochondria were detected with codon. Exon 13 is located 8280 base pairs downstream mitotracker (Fig. 1B), and nuclei were detected with of exon 12 and 4542 base pairs upstream of exon 14, DAPI staining (Fig. 1A,B). Confocal microscopy ana- former exon 13, in the genomic sequence of the VRK2 lysis showed that a significant fraction of the endog- gene (Fig. 2A). The resulting VRK2 proteins, A and B, enous protein was membrane bound both to the ER have 508 and 397 amino acids, respectively. They differ and, to a lesser extent, to mitochondria, as detected by in the C-terminus. In isoform A, this region (residues the overlapping signals. The data suggested that 395–508) contains a hydrophobic sequence (residues

Fig. 1. Subcellular localization of the endo- genous human VRK2 protein in MCF7 and WSI cell lines. VRK2-specific detection was determined with a rabbit polyclonal anti- body. DAPI staining, used to identify the nuclei, is also shown. The ER was identified with a monoclonal antibody specific for caln- exin (A). Mitochondria were detected using the MitoTracker Red CMXRos reagent (B). Bars ¼ 50 lm.

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2489 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al.

Fig. 2. Generation of two VRK2 messages by alternative splicing. (A) Detection of the new exon identified in the message coding for the VRK2B isoform. The DNA sequences correspond to the genomic sequence (Ensembl genomic location: AC068193.7.1.170059), and the cDNA for VRK2A (AB000450) and VRK2B (AJ512204). (B) Alignment of the VRK2A and VRK2B protein sequence to show the divergent C-terminus, the location of catalytic region and other specific features. The arrow indicates where the reading frame changed as a consequence of alternative splicing.

487–506). In isoform B, this region is replaced by three lines, but VRK2B protein was more abundant in some amino acids (VEA), however, the catalytic domain at cell lines, such as C4-I, HeLa, MCF7 or the colon the N-terminus is identical (Fig. 2B). carcinoma WiDr, and detected in smaller amounts in the remaining carcinoma cell lines (Fig. 3A). In lym- phoma cell lines, only isoform A was detected. To Differential expression of VRK2A and VRK2B identify the mobility of each protein, protein extract proteins in tumour cell lines of MCF7 cells was run in parallel with the purified To demonstrate that the two messages identified code VRK2A and VRK2B proteins expressed as glutathi- for real proteins, we determined their presence in a one S-transferase (GST)–fusion proteins and digested panel of tumour cell lines using western blot analysis, with thrombin. We observed identical mobility in the with a polyclonal antibody raised against full-length endogenous proteins with the cloned and purified iso- VRK2 protein (isoform A), but that recognizes the forms (Fig. 3B). N-terminal region common to both isoforms (not The relative concentration of mRNA was also deter- shown). The VRK2A protein was detected in all cell mined by real-time quantitative PCR in the H1299 and

2490 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms

Fig. 3. Expression of two VRK2 isoforms. (A) Detection of VRK2 proteins in several cell lines that were determined by immunoblotting of whole-cell extracts. Whole extract from each cell line was fractionated in an SDS-polyacrylamide gel and transferred to a poly(vinylidene difluoride) membrane. The blot was developed with a specific polyclonal antibody that detects both VRK2 isoforms. The cell lines proceed from different types of tumours as indicated in the figure and include carcinomas of different types (squamous and adenocarcinomas), sarco- mas and T and B lymphomas. (B) The mobility of endogenous VRK2A and VRK2B proteins from MCF7 cell extracts was compared with bac- terially expressed and purified GST–VRK2A and GST–VRK2B proteins that were digested with thrombin. The VRK2 isoforms were detected in the western blot with a rabbit VRK2-specific polyclonal antibody. (C) Quantitative detection by real time RT-PCR of total VRK2 (isoform A plus B) messages (solid lines) or specific VRK2B message (dotted lines) in the H1299 (blue) and MCF7 (pink) cell lines. (D) Localization of endogenous VRK1 and VRK2 proteins in three adenocarcinoma cell lines. VRK1 was detected with a mouse monoclonal antibody specific for human VRK1. Human VRK2 was detected with a rabbit polyclonal antibody. Bar ¼ 50 lm.

MCF7 cell lines. In this experiment, total and specific Therefore, the localization of endogenous VRK1 and VRK2B messages were detected. In both cases there VRK2 proteins was simultaneously determined by con- was always more VRK2A than VRK2B mRNA, and focal microscopy in MCF7 cells from a breast adeno- the two cell lines appeared to have similar levels of carcinoma, A549 cells from lung adenocarcinoma and each message (Fig. 3C). HeLa cells from a cervical adenocarcinoma. Immuno- The nuclear localization of VRK2 suggests that it fluorescence showed that VRK1 is cytosolic with a might be redundant, with VRK1 reported to be exclu- particulate aspect in these three cell lines, whereas sively nuclear in transfection experiments [11,13,15]. VRK2 is located in both the cytoplasm and is clearly However, the nuclear localization of VRK1 is depend- detected in the nucleus (Fig. 3D). In MCF7 and HeLa ent on the cell type, in lymphomas, sarcomas and cells the strong staining in the nucleus coincides with squamous carcinomas it is nuclear, but in some adeno- the more abundant expression of VRK2B detected by carcinomas it is cytosolic (manuscript in preparation). western blot.

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2491 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al.

The two VRK2 isoforms have a different Substrate specificity of VRK2 isoforms and p53 subcellular localization phosphorylation To identify and confirm the subcellular localization of Despite the C-terminal domain differences, the cata- both VRK2 isoforms the cDNA of each isoform was lytic domains are identical in both VRK2 isoforms. To cloned in pCEFL–HA vector that contains an HA epi- determine if there was any difference in substrate spe- tope tag (because there is no monoclonal antibody spe- cificity between the two isoforms, a panel of substrates cific for each isoform), resulting in clones pCEFL– commonly used to characterize Ser-Thr kinases related HA–VRK2A and pCEFL–HA–VRK2B. The presence to casein kinase I were used [1,11]. Both isoforms of a hydrophobic tail in VRK2A suggests that this iso- phosphorylated casein, histone 2B and myelin basic form might be associated with membranes. Cos1 cells protein in a similar manner, but did not phosphorylate were transfected with each of the constructs and the histone 3 (not shown). Furthermore, the two VRK2 location of the transfected protein was determined with isoforms had a strong autophosphorylation activity an anti-HA serum. Isoform VRK2A was localized to in vitro when tested as GST–VRK2 fusion proteins the membrane of the ER and nuclear envelope, as (Fig. 5A). shown by its colocalization with calreticulin (Fig. 4A). To identify substrates of the VRK2 kinase that are Isoform VRK2B, lacking the transmembrane of biological relevance we analysed the effect on the domain, presented a diffuse pattern throughout the phosphorylation of p53 in its N-terminus, transactiva- cytoplasm and some was even detected in the nucleus tion domain, because it is a known substrate of its and outside the nucleolus. Because of this nuclear pres- related kinase VRK1 [11]. For this we used several ence we tested whether VRK2B shares a subcellular GST–p53 (murine) fusion proteins containing individ- location with a known target of the related VRK1 pro- ual or combined substitutions of Ser or Thr residues tein, such as the p53 tumour-suppressor protein [11,12]. [61,62]. There was a loss of radioactive signal whenever For this, H1299 (p53– ⁄ –) cells were cotransfected with the Thr18Ala substitution was introduced by itself or either of the VRK2 isoforms and pCB6 + p53. in combination with Ser15Ile or Ser20Ala. VRK2A VRK2B, but not VRK2A, and p53 proteins were detec- and VRK2B have an identical in vitro phosphorylation ted in the nucleus, with some overlap in their fluores- pattern (Fig. 5A), and Thr18 appears to be the cence, and outside the nucleolus (Fig. 4B). main residue phosphorylated, as detected by a loss of

Fig. 4. Subcellular localization of VRK2A and VRK2B proteins. Cos1 or H1299 cell lines were transfected with either constructs of VRK2A or -B tagged with the HA epitope and expressed in a pCEFL vector. Expres- sion of VRK2 isoforms was detected with a monoclonal antibody specific for the HA epi- tope. (A) Colocalization of HA–VRK2A with calreticulin, a marker for the ER, was detec- ted with an anti-calreticulin serum in Cos1 cells. Nuclei were identified with DAPI. (B) Colocalization in the nucleus of H1299 cells of transfected HA–VRK2B and p53. The HA–VRK2B protein was detected with an antibody against the HA epitope tag. The p53 protein was detected with a mix of DO1 and Pab1801 monoclonal antibodies. Bar ¼ 50 lm.

2492 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms

Fig. 5. Phosphorylation of p53 in vitro by VRK2A and VRK2B. (A) Phosphorylation of GST–p53 (murine) fusion proteins with different individ- ual amino acid substitutions by the VRK2A (upper) and VRK2B (lower) isoforms. The GST–p53 substrates used were FP221, residues 1–85; FP279, residues 11–65; FP267, residues 1–64. Fusion proteins were made with the murine p53, but the numbering is that of the human p53 protein in this conserved region. Individual Ser or Thr substitutions are indicated. (B) Phosphoamino acid analysis of phosphorylated GST–p53, the staining with ninhydrin and the radioactivity incorporation are shown. (C) Phosphorylation of human p53 by VRK2B. Four human GST–p53 constructs spanning different regions of p53 were used as substrates of VRK2B. On the left is shown the detection of the proteins with Coomassie Brilliant Blue staining and to the right is shown the incorporation of radioactivity. (D) Interaction between VRK2B and p53. H1299 cells were transfected with plasmids pGST–VRK2B or kinase-dead pGST–VRK2B(K169E) and pCB6 + p53 or pCB6 + p53T18A in the combinations indicated in the figure and their correct expression was checked in the cell lysate prepared 48 h after transfection (upper). The lysate was mixed with glutathione–Sepharose beads for 4–12 h at 4 C with and the beads were pulled-down by centrifugation. The proteins brought down with the beads were analysed in an immunoblot with specific antibodies (lower). (E) Lack of Hdm2 phosphorylation by the VRK2B isoform. As substrates VRK2B kinase two different Hdm2 proteins were used; a full-length protein with a His tag and a GST fusion protein of the Hdm2 amino terminus (residues 1–188). In the left panel is shown the Coomassie Brilliant Blue staining and in the right-hand panel is shown the incorporation of radioactivity.

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2493 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al. phosphate incorporation. Similar results were obtained results in disruption of this interaction and conse- using a construct spanning p53 residues 1–85, 1–64 or quently in its stabilization [63]. Therefore, we deter- 11–63. Phosphorylated GST–p53 was used to deter- mined using western blot analysis whether the mine the incorporation of radioactivity in a phospho- phosphorylation of p53 by VRK2B or VRK2A also amino acid analysis. Incorporation was seen only in resulted in its stabilization. H1299 (p53– ⁄ –) cells were threonine residues, which is unique in the common transfected with pCB6 + p53 and increasing amounts region (Fig. 5B). of pCEFL–HA–VRK2A or pCEFL–HA–VRK2B. Next, the phosphorylation of human p53 was stud- VRK2B, but not VRK2A, induced an accumulation of ied using either a full-length protein or partial p53, which was higher as the amount of transfected constructs. Phosphorylation was detected only in con- VRK2B was increased (Fig. 6). To confirm that both structs within the N-terminal p53 region, as expected (Fig. 5C), and no incorporation was detected associ- ated with constructs spanning residues 90–390. In kinase reactions the interaction between the kin- ase and substrate is very short. However, it is some- times possible to detect stable intermediate complexes between a kinase and its substrate if the reaction can- not be performed. Therefore, to address the possibility that VRK2B might form a complex with p53 an experiment using different proteins, either wild-type or mutated, was designed. H1299 cells were transfected with pGST–VRK2B mammalian expression constructs, both the wild-type and the inactive kinase (pGST– VRK2B–K169E), which express a catalytically inactive form of VRK2B because it lacks the lysine essential for kinase activity. As a substrate we used p53 or its nonphosphorylatable p53T18A variant. Expression of the different proteins was confirmed in whole-cell lysates (Fig. 5D, upper), and these lysates were used for a pull-down of GST–VRK2B-associated proteins identified by immunoblot analysis. Formation of a sta- ble complex was detected when an inactive kinase VRK2B(K169E) was used, independent of the p53 sta- tus, either wild-type or mutated p53T18A. No stable complex could be formed between an active VRK2B and a nonphosphorylatable p53. The inability of an inactive kinase to transfer the phosphate resulted in the formation and detection of a stable intermediate VRK2B(K169E)–p53 complex (Fig. 5D, lower). Because the N-terminus of p53 interacts with Mdm2, the potential phosphorylation of Hdm2 (human Mdm2) was studied using either full-length protein or its N-terminus (residues 1–188), which is the region of interaction with p53, but we did not detect Fig. 6. Stabilization of transfected p53 induced by VRK2B. H1299 any phosphorylation by VRK2A or VRK2B isoforms (p53– ⁄ –) lung carcinoma cells were transfected with 25 ng of (Fig. 5E; VRK2A not shown). pCB6 + p53, and varying amounts (0, 2, 3 and 4 lg) of plasmids expressing pCEF–-HA–VRK2A, pCEF–-HA–VRK2B or the kinase dead pCEF–-HA–VRK2B(K169E). Detection of total p53 from in VRK2B but not VRK2A induces the accumulation whole-cell lysate extracts was carried out with a mix of DO1 and of p53 Pab1801 antibodies. At the bottom is shown the quantification of p53 normalized with b-actin depending on the kinase isoform used The p53–Mdm2 interaction promotes p53 ubiquitina- in the assay as the mean of two independent experiments. West- tion and degradation via the proteasome pathway. ern blots were performed using the specific antibodies indicated in Phosphorylation of p53 at its N-terminus frequently Experimental procedures.

2494 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms

HA-tagged VRK2 isoforms expressed in culture were active, an in vitro kinase assay with the immunoprecip- itated protein was performed (data not shown). There- fore, p53 in vivo is one target of VRK2B, probably because they colocalize in the same compartment. However, VRK2A, which is in the cytosol in a mem- brane-bound form, is not able to induce p53 accumula- tion. To confirm that this was dependent on the activity of VRK2B, we used a construct pCEFL–HA– VRK2B(K169E) that expresses a catalytically kinase dead form of VRK2B because it lacks the lysine essen- tial for kinase activity. Indeed, this inactive mutant of VRK2B did not induce accumulation of p53 (Fig. 6).

The stabilization of p53 by VRK2B is a post-translational effect To determine whether the effect induced by VRK2B on the p53 levels is a consequence of post-translational modification, an experiment was performed in the presence of the protein synthesis inhibitor cyclohexi- Fig. 7. Stability of p53 by VRK2B is a post-translational effect. mide. H1299 cells were transfected with pCB6 + p53 H1299 cells (p53– ⁄ –) were transfected with 50 ng of pCB6 + p53, or a combination of pCB6 + p53 and pCEFL–HA– without (upper) or with (middle) 4 lg of pCEF–-HA–VRK2B, 36 h VRK2A or pCEFL–HA–VRK2B, 36 h later cyclohexi- after transfection cycloheximide (CHX) was added to the culture at a final concentration of 60 lgÆmL)1 and the levels of p53 were mide was added and the levels of p53 were determined determined at different time points by immunoblot analysis. Cell by immunoblots at different time points. The amount lysates were analysed by western blot. The p53 protein was detec- of protein was normalized to the level of b-actin. In ted with a mix of DO1 and Pab1801 antibodies, and HA–VRK2B the absence of VRK2B, p53 was degraded almost was detected with anti-HA serum. At the bottom it is also shown immediately, as expected, whereas its levels were stable the relative level of p53 normalized with the b-actin level at each for more than 8 h in the presence of VRK2B (Fig. 7). time point. C represents a control for transfection with an empty A similar experiment was performed with VRK2A; vector without p53. this isoform did not protect p53 from degradation. reporter containing the Mdm2 promoter. VRK2B also activated the transcription of Mdm2 (Fig. 8B). Next, VRK2B stabilizes endogenous p53 and activates A549 cells were transfected with increasing amounts of its transcriptional activity pCEFL–HA–VRK2B, and the luciferase activity of Next we determined whether VRK2B could also stabil- the p53–Luc reporter was determined. As VRK2B ize the endogenous p53 protein. For this assay the increased, so did endogenous p53-dependent transcrip- A549 cell line (p53+ ⁄ +) was used. After transfection tion (Fig. 8C). with VRK2B, cells were treated with cycloheximide, and the level of endogenous p53 protein was deter- p53 phosphorylation by VRK2B differs from that mined at different times. The stability of endogenous induced by adriamycin or taxol p53 (half-life) was less than 20 minutes in the absence of VRK2B, but in its presence, the stability of p53 was We showed that VRK2B phosphorylates p53 in vivo at significantly increased (Fig. 8A). A consequence of this Thr18. First it was confirmed that transfected p53 was stabilization by VRK2B is that p53 might be transcrip- phosphorylated at Thr18 by VRK2B in H1299 cells tionally active. To determine whether the VRK2 pro- (p53– ⁄ –). When the cells were transfected with VRK2B, teins affected transcription, A549 cells were transfected there was an increase in p53 phosphorylation as shown with a p53 synthetic reporter, plasmid p53–Luc, which by western blot analysis (Fig. 9A, upper). However, contains several p53-response elements. VRK2B, but because only a fraction of the cells were transfected not VRK2A, activated this promoter (Fig. 8B). In and the p53-Thr18 phospho-specific antibody is not order to verify whether VRK2B affected the transcrip- very sensitive, the specific signal is difficult to detect tion of a p53 target gene we used the pMdm2–Luc in whole-cell extracts. To improve detection, p53 was

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2495 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al.

Fig. 8. Stabilization of endogenous p53 and activation of p53- dependent transcription. (A) Stabilization of endogenous p53 by the VRK2B isoform in A549 (p53+ ⁄ +) lung carcinoma cell line. A549 cells were transfected with 4 lg of pCEF–-HA–VRK2B and 36 h after transfection cycloheximide was added to the culture. The amount of endogenous p53 protein at different time points was determined with a mix of DO1 and Pab1801 antibodies and HA–VRK2B was determined with anti-HA serum. (Upper) Levels of p53 in the absence of VRK2B, (lower) levels of p53 in the presence of VRK2B. (B) Activation of endogenous p53-dependent transcrip- tional activity by VRK2B. A549 cells were cotransfected with or without VRK2 isoforms and a p53 synthetic reporter plasmid (p53– Luc, containing p53 response elements) or a specific gene promo- ter (Mdm2–Luc,containing Mdm2 promoter). pBASIC and pSV40- Control reporters were used, respectively, as negative and positive controls of the luciferase assay. Reporter luciferase activity was normalized for transfection levels with Renilla luciferasa (pRL-tk). (C) Dependence of transcriptional activation on the dose of trans- fected VRK2B. A549 cells were transfected with increasing amounts of pCEF–-HA–VRK2B and 0.5 lg of p53–Luc reporter con- struct, and 0.3 lg of pRL-tk Luc. Luciferase activity was deter- mined 48 h after transfection and normalized with Renilla luciferase activity. Data from at least three independent experiments were analysed with Student’s t-test. *P < 0.05; **P<0.005.

VRK2B. In the same experiment the phosphorylation of Ser15 was also determined. However, the relative phosphorylation of Thr18 was higher in the presence of VRK2B than in the presence of adriamycin, and was similar to that induced by taxol (Fig. 9B, middle). VRK2B induced relatively higher phosphorylation of Thr18 with respect to Ser15 phosphorylation, while in the presence of taxol or adriamycin, both residues were phosphorylated equally (Fig. 9B, lower), suggesting that VRK2B did not require previous phosphorylation on Ser15. The higher absolute signal in drug-treated cells is due to the fact that all cells responded, whereas only a fraction were transfected with the VRK construct.

VRK2B reduces p53 ubiquitination and promotes concentrated by immunoprecipitation, and phosphory- its acetylation by p300 lation at Thr18 was determined using a phosphor- specific antibody. Inclusion of VRK2B increased the Phosphorylation of p53 at its N-terminal region modu- phosphorylation of transfected p53 in Thr18 by lates its affinity for different binding proteins. There- approximately fourfold, as shown in Fig. 9A (lower). fore, a consequence of p53 phosphorylation at Thr18 The next step was to determine the phosphorylation might be to change its binding characteristics to of endogenous p53 protein in A549 cells (p53+ ⁄ +)in Mdm2 [58,64,65]. Based on the phosphorylated residue the presence of overexpressed VRK2B; as a positive by VRK2B, the expected effect would be a reduction control for phosphorylation, cells were treated with in the p53–Mdm2 interaction [54], and consequently a either adriamycin or taxol. All, overexpression of decrease in its ubiquitination. In H1299 cells, transfec- VRK2B or drugs induced Thr18 phosphorylation as tion of VRK2B clearly reduces the ubiquitination of detected using a phosphor-specific antibody (Fig. 9B). p53 by exogenous Mdm2 (Fig. 10A), indicating that Thr18 phosphorylation was dependent on the dose of the phosphorylation induced by VRK2B might alter

2496 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms

Fig. 9. In vivo phosphorylation of p53 Thr18 by VRK2B. (A) Phosphorylation of transfect- ed p53 in H1299 cells (p53– ⁄ –) by VRK2B. H1299 cells were transfected with 0.1 lgof pCB6 + p53 and 6 lg of pCEF–-HA–VRK2B. Thirty-six hours after transfection the cells were lysed for western blot analysis (upper) or immunoprecipitation of p53 followed by western blot (lower). The quantification of the relative phosphorylation of p53 is shown. (B) Phosphorylation of endogenous p53 in Thr18 by VRK2B in A549 cells (p53+ ⁄ +). As positive control treatment with adriamycin (0.5 lgÆmL)1 for 8 h) or taxol (100 nM for 12 h) are included. VRK2B phos- phorylates p53 in Thr18 in a dose-dependent manner. Adryamicin and taxol treatments also increase Thr18 phosphorylation. The middle graph shows relative Thr18 phos- phorylation, and the lower graph shows Thr18 phosphorylation with respect to Ser15 phosphorylation. the interaction of p53 and Mdm2, therefore causing lular location. VRK2A localizes to the ER, whereas p53 accumulation. VRK2B variant, in contrast to those previously detec- The coactivator p300 also interacts with the p53 ted by RT-PCR [13], is detected in both the cytoplasm transactivation domain and contributes to p53 stabil- and nucleus, outside the nucleolus, despite lacking a ization and activation. To test the possibility that known NLS. VRK2A is highly expressed in different phosphorylated p53 might bind to this transcriptional cell lines, whereas VRK2B expression is more restric- coactivator, H1299 cells were transfected with different ted and appears to be coordinated by an unknown combinations of VRK2B (pCEFL–HA–VRK2B), p53 mechanism. Because different cell types present differ- (pCB6 + p53) and p300 (pHA-p300) expression con- ent proportions of both isoforms, and the substrate structs. The effect on p53 protein levels and degree of specificities of both VRK2 isoforms and their relative acetylation was determined by immunoblot analysis, VRK1 [11,16,17] are similar in vitro, we postulate that with an antibody specific for acetylation in Lys373 and the biological effects mediated by these kinases might Lys382 of whole-cell extracts. Indeed, it was observed be determined by their subcellular localization and the that VRK2B facilitates p53 acetylation (Fig. 10B). The proteins present in the corresponding compartment, p53 protein was also concentrated by immunoprecipi- and by their C-terminal domain, which mediates inter- tation to improve detection of its acetylation. In the actions with different proteins. Their regulation is absence of p300, the acetylation of p53 was enhanced likely to be controlled by the different C-terminus, slightly by VRK2B, and was particularly noticeable which determines their interaction with other regula- when cotransfected with pCMV-p300-HA (Fig. 10C), tory proteins not yet identified, but whose future iden- indicating that p53 stabilized by VRK2B was acetylated tification will contribute to the unravelling of the de novo. signalling pathway to which these kinases belong. For instance, VRK2B and VRK1 are localized in the nucleus but differ in their last 90 amino acids in the Discussion C-terminus. Alternative splicing of the human VRK2 gene gener- Stabilization of p53 is part of the response to cellu- ates two isoforms with a common kinase domain and lar stress, and is usually mediated by phosphorylations a different C-terminus, which determines their subcel- carried out by a large number of kinases that target

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2497 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al.

Fig. 10. Effect of VRK2B on ubiquitination and acetylation of p53. (A) Reduced ubiquitination of p53 by Mdm2 in the presence of VRK2B. H1299 cells (p53– ⁄ –) were cotransfected with 0.5 lg of pCB6 + p53, 0.5 lg of BRR12-His-ubiquitin, 1.5 lg of pCOC-Mdm2-X2 and 2.5 lgof pCEF–-HA–VRK2B. Transfections were performed with the JetPEI reagent. 36 h after transfection 5 lM lactacystin (a proteasome inhibitor) was added to the culture and 12 h later the cells were lysed with RIPA buffer for analysis by western blot. Single p53 and ubiquitinated p53 were detected with a mix of DO1 and Pab1801 antibodies, HA–VRK2B was detected with anti-HA monoclonal serum and for Mdm2 detec- tion we used anti-SMP14 monoclonal serum. The bar graph represents the mean of three independent experiments with its SD. *P < 0.02. (B) Increased acetylation of p53 by p300 in the presence of VRK2B. H1299 cells were transfected with the indicated constructs and whole cell extracts were immunoblotted with specific antibodies as indicated. (C) Identification of p53 acetylation promoted by VRK2B. H1299 cells were transfected with the plasmids indicated and p53 was immunoprecipitated with a mix of DO1 and Pab240 antibodies. The cells were treated with 5 lM of Trichostatin A from Streptomyces sp. (TSA), a deacetylase inhibitor. Cells were lysed in the acetylation-precipitation buf- fer with TSA, and p53 was immunoprecipitated with a mix of DO1 and Pab240 antibodies. The acetylation of p53 in residues Lys373 and Lys382 was determined with a specific antibody as indicated in Experimental procedures. The constructs used in transfections were pCB6 + p53, p-CEFL-HA–VRK2B and pCMV-p300-HA. The bar graph represents the mean of two independent experiments with its SD and *P < 0.05. (D) H1299 cells were transfected with plasmid encoding wild-type p53 or different substitution as indicated, with or without pCEF–-HA–VRK2B. Detection of total p53 from in whole-cell lysate extracts was carried out with a mix of DO1 and Pab1801 antibodies. several different residues, mainly located in the p53 tion is mediated by some kinases, of these casein kin- N-terminus, and generates a complex pattern of phos- ase 1 delta requires the previous phosphorylation of phorylation [33]. Furthermore, the p53 N-terminus Ser15 [50] and is cytosolic in interphase [51], whereas mediates the interaction using many regulatory mole- VRK1 or VRK2B phosphorylate Thr18 directly and cules such as acetyl transferases [58] and ubiquitin are nuclear proteins [11]. Thr18 phosphorylation con- ligases, for example, its downregulator Mdm2 [66]. tributes to p53 stabilization. The other VRK2A, which The specificity of the interactions can be modulated by has a different subcellular location and does not colo- phosphorylation at different residues, of which Ser15 calize with p53, is not able to induce stabilization. and Ser20 are the best characterized, and result in p53 Cytosolic VRK2A is therefore more like casein kin- stabilization [41]. However, stabilization can also occur ase 1 delta as both phosphorylate Thr18 in vitro [50]. in the absence of Ser15 or Ser20 phosphorylation [46], Therefore, a different subcellular location and different in this case, Thr18 phosphorylation is the other likely modulation of a common kinase domain may deter- mechanism for p53 stabilization. Thr18 phosphoryla- mine different biological activities. Phosphorylation of

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Thr18 has been observed in vivo in cells treated with proteins, one is the coordination of their expression, taxol [59,67] and in senescent fibroblast [60]. However, VRK2B is formed when VRK1 is cytosolic, but not the phosphorylation at Thr18 induced by taxol or when is nuclear; and the other is how VRK2B is trans- adriamycin is also accompanied by the phosphoryla- ported to the nucleus because it lacks a nuclear local- tion of Ser15, probably via activation of stress- ization signal, that is present in the C-terminus of response pathways; this is not the case for VRK2B, VRK1 [11]. The effect of VRK2B on p53 Thr18 phos- which is probably mediated via a different signalling phorylation is functionally redundant with the effect of pathway or cooperates with some of these stress- VRK1 [15] regarding p53. VRK2B can therefore func- response pathways. It has also been postulated that tionally replace VRK1 in those cells where the latter is Thr18 phosphorylation might play a role in normal cytosolic, although they are likely to be regulated dif- growth [15], and this may be the reason for its detec- ferently, because they differ in their C-terminus. This tion in cellular senescence [60]. function would suggest that there should be some Phosphorylation of the N-terminus might be a regu- redundancy in these genes, and this would explain why latory mechanism for a docking site in p53, partic- the murine knockout of VRK2 is viable [71,72]. The ularly in Thr18, which affects the interaction with VRK family of Ser-Thr kinases represents the begin- either Mdm2 [54] or p300 [58,64,68]. The consequence ning of novel signalling pathways that operate in many of p53T18 phosphorylation is a disruption of p53 cell types and that are beginning to be unravelled. interaction with Mdm2, because this residue is essential for maintaining the structure of the p53-binding region Experimental procedures [53,54,69], by reducing p53 ubiquitination it contri- butes to its stabilization and promotes other protein RNA extraction, RT-PCR, cloning and interactions [15]. The coactivator p300 binds to p53 in mutagenesis the same region as Mdm2, and promotes its stabiliza- tion and transactivation by acetylation [70]. In the case Total RNA from H1299, HeLa and MCF7 was isolated of Thr18 phosphorylation by VRK2B, there is an using RNAeasy Mini Kit (Qiagen, Hilden, Germany). increase in p53 stability and transactivation, as expec- RNA was analysed and quantified using a Bioanalyser 2100 ted, if improved binding to p300 and disruption of the nano-lab chip from Agilent Technologies (Waldbronn, Mdm2 interaction were promoted by this phosphoryla- Germany). cDNA was synthesized as previously reported tion [55,58]. The effects observed regarding phosphory- [11]. Different primers were designed to generate full-length lation of human p53 in Thr18 are consistent with VRK2 (nucleotides 130–1657; GenBank accession number similar findings reported for the role of the equivalent AB000450) based on human VRK2 [7]. VRK2A: 5¢- residue in murine p53, Thr21 [48], and it has also been CCCGGATCCATGCCACCAAAAAGAAATGAAAAAT reported that Thr18 phosphorylation by VRK1 stabil- ACAAACTTCC-3¢ (nucleotides 130–165), this primer has the initiation codon and a BamH1 cloning site. VRK2X: 5¢- izes p53 and promotes its binding to Mdm4 and p300 GGGTCTAGAGGAATTTTGGTATCATCTTCAGAG-3¢ [15]. Although p53 can be phosphorylated at several (nucleotides 1652–1675 antisense strand), with an XbaI clo- residues by many kinases, the precise functional conse- ning site and the termination codon. VRK2S: 5¢-GGGGTC quences resulting from different combinations of phos- GACGGAATTTTGGTATCATCTTCAGAG-3¢ (nucleo- phorylated residues are not well known. tides 1652–1675 antisense strand), with a SalI cloning site It has been proposed that the nuclear kinase VRK1 and the termination codon. Human full-length VRK2 was has a role in the regulation of p53 in the cellular made with primers VRK2A and VRK2X or VRK2S from response to suboptimal stress during interphase [15], HeLa RNA for cloning in pGEX4T-1 (Amersham Bio- which is the normal physiological situation. Phos- sciences, Little Chalfont, UK). For expression in eukaryotic phorylation of Thr18 is likely to be a component of a cells, the VRK2A and VRK2B full-length cDNA were sub- p53 cycle that is necessary to maintain basal levels of cloned in vector pCEFL–HA or pCEFL–GST with restric- p53, these levels are needed if normal proliferation is tion sites BamHI–NotI, using primers VRK2A, to be able to respond to severe stress signals [15]. VRK2Not1.A: 5¢-GGGCGGCCGCGGAATTTTGGTATC However, in adenocarcinoma tumour cell lines, VRK1 ATCTTCAGAG-3¢ and VRK2TN.1: 5¢-GCGGCCGCCTA is localized in the cytoplasm, therefore it may not regu- AGCTTCTACCTGAGCTGCTTC-3¢. To detect the new late p53. In this situation, the VRK2B isoform is exon in VRK2 transcripts two pairs of flanking primers expressed and is located in the nucleus where it might were designed. Forward primer VRK2TA: 5¢-AGTGAGG functionally replace VRK1 in this suboptimal protect- AAGCGCTGAGTCCT-3¢, and reverse primer: VRK2TB: ive role, given the similarity of their effect on p53. 5¢-CAAGAGTCTCAAGAACCTTTG-3¢ which amplify This indicates two novel levels of regulation of VRK fragments of nucleotides 135–179 specific for isoform A

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2499 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al. and B messages, respectively, and forward primer Acid hydrolysis and phosphoamino acid analysis VRK2TC: 5¢-GCAGCTCAGGTAGAAGCTTAGG-3¢, and reverse primer: VRK2TD: 5¢-CGTGCTGACTGTGGAAG An in vitro kinase assay was performed with GST–VRK2B TGT-3¢ specific for isoform B. One hundred nanograms of and GST–p53 (FP267) as substrates. The phosphorylated total RNA was used in a one-step RT real-time PCR proteins were fractionated by SDS ⁄ PAGE and transferred amplification reaction using the ‘Quantitec SYBR Green onto an Immobilon-P filter (Millipore, Bedford, MA), RT-PCR kit’ from Quiagen in an iCycler (Bio-Rad, Hercu- radioactive bands corresponding to GST–p53 were excised les, CA). The reaction was analysed using icycler software and the protein was eluted. The eluted protein was hydro- (Bio-Rad). The VRK2B cDNA sequence has GenBank lysed with 200 lLof6n HCl at 110 C for 1 h. The sam- accession number AJ512204. An inactive or kinase-dead ple was freeze-dried and resuspended in a mixture (KD) VRK2 mutation was made by site-specific mutagen- containing 2 lL of a stock solution at 1 mgÆmL)1 of P-Ser, esis introducing the K169E substitution in mammalian P-Thr and P-Tyr as reference markers, 1 lL of 1% bromo- expression constructs and in GST fusion proteins. Site- phenol blue and 2 lL of formic acid ⁄ acetic acid ⁄ water directed mutagenesis was performed with the Quick-Change (1:3.1:35 v ⁄ v ⁄ v) pH 1.9. The mixture was applied to a cellu- Mutagenesis kit from Stratagene (San Diego, CA). lose chromatoplaque (Merck) and subjected to flat-bed electrophoresis in a Multiphor II system (Amersham Bio- sciences) at 1.5 kV for 90 min. After air drying, the radio- Plasmids and fusion proteins activity on the chromatoplaque was exposed to X-ray film. The GST–p53 constructs containing the N-terminus of Phosphoamino acid markers were detected by staining with murine p53 and its mutants were a gift of D. Meek (Dundee a 0.25% ninhydrin solution in acetone. University, UK) [61,62]. The human GST–p53 fusion pro- teins, 1–390 (full-length), 90–290 and 290–390 were from T. Cell lines, transfections and luciferase reporter Kouzaridis (Cancer Research UK, Cambridge, UK). Full- assay length human pHdm2-his and N-terminus of pGST–Hdm2 (residues 1–188) were from A. Levine (Rockfeller University, Cell lines 293T, C4-I, HeLa, Siha, SW756, Cal51, H9528, New York). GST-fusion proteins were expressed in A498, HepG2, Cos1, Colo 320, HCT116, OSA, RMS13, BL21DE3 cells and purified using glutathione–Sepharose NIH 3T3 and WiDr were grown in DMEM; and cell lines (Amersham) as previously reported [11]. His-tagged proteins Jukat, JY, HPBALL, Jijoye, HL60, H1299, A549 and were expressed in, purified with TALON Metal Affinity MCF7 were grown in RPMI both supplemented with 10% Resin (BD Biosciences, Erembodegem, Belgium) and eluted fetal bovine serum, glutamine, penicillin, and streptomycin with 100 nm imidazol (Merck, Darmstadt, Germany). Mam- in a humidified 5% CO2 atmosphere. For H1299 and A549 malian expression plasmids containing human p53 wild-type cells, 0.5 · 106 cells were plated in 60 mm dishes, or cDNA, pCB6 + p53, and pCOC-Mdm2-X2 were from K. 1 · 106 in 100 mm diameter dishes. After 24 h the cells Vousden (Beatson Institute, UK) and plasmid pCMVb-p300- were transfected with the indicated amounts of plasmids HA was from R. Eckner (University of Zurich). The ubiqu- and JetPEI reagent (PolyTransfection, Illkirch, France) itin tagged with a His epitope for mammalian expression according to the manufacturer’s recommendations. Empty (clone BRR12-His-ubiquitin) was from S. Lain and D. Lane vector pGEX4T1 was used as carrier. In the experiments (Dundee University, UK). Plasmid pMdm2–Luc was a gift determining p53 accumulation were used 25 ng of from B. Vogelstein. The p53-cis reporter plasmid (p53–Luc) pCB6 + p53 and increasing amounts of pCEF–-HA– with the luciferase gene was from Stratagene (La Jolla, CA). VRK2A or pCEF–-HA–VRK2B. Cycloheximide (60 lgmL)1) was added 36 h after transfection. Adriamy- cin (Sigma, St. Luis, MO) was added to a final concentra- Ser-Thr kinase activity assay ) tion of 0.5 lgmL 1 and Taxol was used at 100 nm. For the Kinase activity was determined by assaying protein phos- ubiquitination assay 0.5 · 106 H1299 cells were seeded in phorylation in a mixture containing 20 mm Tris ⁄ HCl, 60 mm dishes and transfected with 0,5 lg of pCB6 + p53, pH 7.5, 5 mm MgCl2, 0.5 mm dithiothreitol, 150 mm KCl 0,5 lg of BRR12-His-ubiquitin, 1,5 lg of pCOC-Mdm2-X2 and 5 lm (5 lCi) [32P]ATP[cP] (Amersham) with 2 lgof and 2,5 lg de pCEF–-HA–VRK2B with 10 lL of JetPEI GST–VRK2 protein and 10 lg of another protein as sub- reagent and 36 h after transfection, the proteasome inhib- strate when indicated. The reaction was routinely per- itor lactacystin (Sigma) was used at 5 lm. For the acetyla- formed for 30 min at 30 C [11]. The phosphorylated tion assay 1 · 106 H1299 cells were plated in 100 mm products were analysed in an SDS-polyacrylamide gel and diameter dishes and were transfected with 0,1 lgof the incorporation of radioactivity was determined either by pCB6 + p53, 5 lg of pCMV-p300-HA and 5 lgde autoradiography or in a Molecular Imager FX (Bio-Rad). pCEF–-HA–VRK2B with 20 lL of JetPEI reagent; 5 lm Phosphoamino acid analysis was performed as previously Trichostatin A was added to the culture 6 h before described [16,17]. harvesting cells. For transcription assay experiments,

2500 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms

0.5 · 106 A549 cells were seeded in 60 mm dishes and 24 h beads were washed three times with lysis buffer and ana- later were transfected in triplicate with JetPEI reagent and lysed by SDS ⁄ PAGE. To detect endogenous VRK2 a rab- 0.5 lg of p53–Luc as luciferase reporter, and increasing bit polyclonal antibody which recognized both isoforms amounts of pCEF–-HA–VRK2A or pCEF–-HA–VRK2B was prepared by immunizing rabbit with human VRK2A and as internal control for transfection we used 0.3 lgof GST-fusion protein. VRK1 was detected with a mouse pRenilla-tk (pRL-tk) reporter plasmid from Promega monoclonal antibody, clone 1F6, prepared against the (Madison, WI). Cells were lysed 48 h after transfection and human VRK1 protein. The monoclonal antibody specific luciferase activity was determined with Dual-luciferase for the HA epitope was from Covance (San Francisco, Reporter Assay System (Promega) in a luminometer CA). The polyclonal antibody specific for calreticulin was Minilumat LB9506 (Berthold, Bad Wildbad, Germany). from Calbiochem (La Jolla, CA) and monoclonal anti- (calnexin AF18) was from Santa Cruz. p53 protein was detected with a mix of DO1 antibody (Santa Cruz) and Immunofluorescence Pab1801 (Santa Cruz) used at 1:500 and 1:1000, respect- Cells were seeded in 60 mm dishes, grown on a coverslip ively. To detect the phosphorylation of p53 in Thr18 and in and transfected as indicated in specific experiments with Ser15 rabbit polyclonal antibody, P-p53 (Thr18)-R, and a pCEF–-HA–VRK2A and -B and also pCB6 + p53 in the monoclonal P-p53 (Ser15)-R from Santa Cruz were used. A H1299 cell line using the calcium phosphate precipitation rabbit antiserum from Upstate Biotechnology (Lake Placid, method; 48 h post transfection the slides were collected and NY) detecting specifically acetylated p53 in Lys373 and 382 fixed with 3% paraformaldehyde for 30 min at room tem- was used. SMP14 monoclonal antibody (Santa Cruz) was perature, treated with 10 mm glycine for 10 min at room used to detect the Mdm2 protein. The monoclonal anti-(b- temperature and then permeabilized with 0.2% Triton X- actin) was from Sigma. Mitochondria were detected using 100 for 30 min at room temperature. The cells were blocked the MitoTracker Red CMXRos reagent (Molecular Probes, with 1% bovine serum albumin (BSA) in NaCl ⁄ Pi for Eugene, OR). The secondary antibodies used from Amer- 30 min at room temperature followed by double immuno- sham were an anti-(mouse-Cy2) (Fluorolink Cy2), anti- staining with the corresponding antibodies. Finally, cells (rabbit-Cy3) (Fluorolink Cy3) and anti-(rabbit-Cy2) were stained with DAPI (Sigma) 1:1000 in NaCl ⁄ Pi for (Fluorolink Cy2) for immunofluorescence, or an anti- 10 min at room temperature, washed with NaCl ⁄ Pi, and (mouse-HRP) or anti-(rabbit-HRP) for western blots. slides were mounted with Gelvatol (Monsanto). The anal- Luminescence in western blot was developed with an ECL ysis was performed with a Zeiss LSM510 confocal micro- kit (Amersham Biosciences). scope. Acknowledgements Western blot analysis, immunoprecipitation, The technical assistance by Virginia Gasco´n is greatly pull-down assays and antibodies appreciated. SB, LK and FMV were supported by fel- Cells were harvested 48 h post transfection and lysed with lowships from Ministerio de Educacio´n y Ciencia, EU

RIPA buffer (150 mm NaCl, 1,5 mm Mg Cl2,10mm NaF, Marie Curie Training Center, and Fundacio´n Ramo´n 100% glycerol, 4 mm EDTA, 1% Triton X-100, 0,1% SDS, Areces and Fundacio´n Cientı´fica AECC, respectively. 50 mm Hepes pH 7,4, protease and phosphatase inhibitors) This work was supported by grants from Ministerio or in acetylation immunoprecipitation buffer (50 mm de Educacio´n y Ciencia (SAF2004-02900), Fondo de Hepes, pH 7.8, 200 mm NaCl, 1% Triton X-100, 10 mm Investigacio´n Sanitaria (FIS-PI02-0585), Fundacio´nde EDTA, 5 mm dithiothreitol, 5 lm trichostatin A and prote- Investigacio´nMe´dica MM, Junta de Castilla y Leo´n ase and phosphatase inhibitors). Fifty micrograms of total (SAN ⁄ SA04 ⁄ 05 and CSI05A05), and Fundacio´n Mem- protein lysate were analysed in a 10% SDS-polyacrylamide oria Samuel Solo´rzano Barruso. gel. Cell lysates were subjected to p53 immunoprecipitation with monoclonal antibodies DO1 and pab240 (Santa Cruz Biotchnology, Santa Cruz, CA) at 4 C overnight, followed References by adsorption to c-Bind (Amersham Biosciences) and ana- 1 Manning G, Whyte DB, Martinez R, Hunter T & lysed in a 7.5% SDS-polyacrylamide gel. For GST pull- Sudarsanam S (2002) The protein kinase complement of down assays cells were lysed with a buffer containing buffer the . Science 298, 1912–1934. 20 mm Tris ⁄ HCl pH 7.4, 137 mm NaCl, 2 mm EDTA, 2 Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, 25 mm b-glycerophosphate, 2 mm pyrophosphate, 10% Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann (v ⁄ v) glycerol and 1% Triton X-100 plus protease inhibi- M et al. (2003) Systematic functional analysis of the tors. GST pull-downs were performed by incubating 1 mg Caenorhabditis elegans genome using RNAi. Nature 421, of total cell extract with glutathione–Sepharose 4B beads 231–237. (Amersham Pharmacia Biotech) for 12 h at 4 C. Sepharose

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3 Morrison DK, Murakami MS & Cleghon V (2000) Pro- ATF2 transcriptional activity by novel phosphorylation tein kinases and phosphatases in the Drosophila genome. on Thr-73 and Ser-62 and cooperates with JNK. J Biol J Cell Biol 150, F57–F62. Chem 279, 27458–27465. 4 Dhillon N & Hoekstra MF (1994) Characterization of 18 Vega FM, Gonzalo P, Gaspar ML & Lazo PA (2003) two protein kinases from Schizosaccharomyces pombe Expression of the VRK (vaccinia-related kinase) gene involved in the regulation of DNA repair. EMBO J 13, family of p53 regulators in murine hematopoietic devel- 2777–2788. opment. FEBS Lett 544, 176–180. 5 Hoekstra MF, Liskay RM, Ou AC, DeMaggio AJ, Bur- 19 Dorrell MI, Aguilar E, Weber C & Friedlander M bee DG & Heffron F (1991) HRR25, a putative protein (2004) Global analysis of the developing kinase from budding yeast: association with repair of postnatal mouse retina. Invest Ophthalmol Vis Sci 45, damaged DNA. Science 253, 1031–1034. 1009–1019. 6 Ho Y, Mason S, Kobayashi R, Hoekstra M & Andrews 20 Santos CR, Rodriguez-Pinilla M, Vega FM, Rodriguez- B (1997) Role of the casein kinase I isoform, Hrr25, Peralto JL, Blanco S, Sevilla A, Valbuena A, Hernandez and the cell cycle-regulatory transcription factor, SBF, T, van Wijnen AJ, Li F et al. (2006) VRK1 signaling in the transcriptional response to DNA damage in pathway in the context of the proliferation phenotype in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 94, head and neck squamous cell carcinoma. Mol Cancer 581–586. Res 4, 177–185. 7 Nezu J, Oku A, Jones MH & Shimane M (1997) Identi- 21 McLean LA, Gathmann I, Capdeville R, Polymeropou- fication of two novel human putative serine ⁄ threonine los MH & Dressman M (2004) Pharmacogenomic ana- kinases, VRK1 and VRK2, with structural similarity to lysis of cytogenetic response in chronic myeloid vaccinia virus B1R kinase. Genomics 45, 327–331. leukemia patients treated with imatinib. Clin Cancer Res 8 Lin S, Chen W & Broyles SS (1992) The vaccinia virus 10, 155–165. B1R gene product is a serine ⁄ threonine protein kinase. 22 Shiio Y, Eisenman RN, Yi EC, Donohoe S, Goodlett J Virol 66, 2717–2723. DR & Aebersold R (2003) Quantitative proteomic ana- 9 Banham AH & Smith GL (1992) Vaccinia virus B1R lysis of chromatin-associated factors. J Am Soc Mass encodes a 34-kDa serine ⁄ threonine protein kinase that Spectrom 14, 696–703. localizes in cytoplasmic factories and is packaged into 23 Tien ES, Gray JP, Peters JM & Vanden Heuvel JP virions. Virology 191, 803–812. (2003) Comprehensive gene expression analysis of per- 10 Rempel RE & Traktman P (1992) Vaccinia virus B1 oxisome proliferator-treated immortalized hepatocytes: kinase: phenotypic analysis of temperature-sensitive identification of peroxisome proliferator-activated recep- mutants and enzymatic characterization of recombinant tor {alpha}-dependent growth regulatory genes. Cancer proteins. J Virol 66, 4413–4426. Res 63, 5767–5780. 11 Lopez-Borges S & Lazo PA (2000) The human vaccinia- 24 Vernell R, Helin K & Muller H (2003) Identification related kinase 1 (VRK1) phosphorylates threonine-18 of target genes of the p16INK4A-pRB-E2F pathway. within the mdm-2 binding site of the p53 tumour sup- J Biol Chem 278, 46124–46137. pressor protein. Oncogene 19, 3656–3664. 25 Boldrick JC, Alizadeh AA, Diehn M, Dudoit S, Liu 12 Barcia R, Lopez-Borges S, Vega FM & Lazo PA (2002) CL, Belcher CE, Botstein D, Staudt LM, Brown PO Kinetic properties of p53 phosphorylation by the human & Relman DA (2002) Stereotyped and specific gene vaccinia-related kinase 1. Arch Biochem Biophys 399, 1–5. expression programs in human innate immune 13 Nichols RJ & Traktman P (2004) Characterization of responses to bacteria. Proc Natl Acad Sci USA 99, three paralogous members of the mammalian vaccinia 972–977. related kinase family. J Biol Chem 279, 7934–7946. 26 Diehn M, Alizadeh AA, Rando OJ, Liu CL, Stankunas 14 Lazo PA, Vega FM & Sevilla A (2005) Vaccinia-related K, Botstein D, Crabtree GR & Brown PO (2002) Geno- kinase-1. AfCS-Nature Molecule Pages 10.1038/ mic expression programs and the integration of the mp.a003025.003001. CD28 costimulatory signal in T cell activation. Proc 15 Vega FM, Sevilla A & Lazo PA (2004) p53 stabilization Natl Acad Sci USA 99, 11796–11801. and accumulation induced by human vaccinia-related 27 Prives C & Hall PA (1999) The p53 pathway. J Pathol kinase 1. Mol Cell Biol 24, 10366–10380. 187, 112–126. 16 Sevilla A, Santos CR, Barcia R, Vega FM & Lazo PA 28 Vousden KH & Lu X (2002) Live or let die: the cell’s (2004) c-Jun phosphorylation by the human vaccinia- response to p53. Nat Rev Cancer 2, 594–604. related kinase 1 (VRK1) and its cooperation with the 29 Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Cri- N-terminal kinase of c-Jun (JNK). Oncogene 23, 8950– ado LM, Klatt P, Flores JM, Weill JC, Blasco MA & 8958. Serrano M (2002) ‘Super p53’ mice exhibit enhanced 17 Sevilla A, Santos CR, Vega FM & Lazo PA (2004) DNA damage response, are tumor resistant and age Human vaccinia-related kinase 1 (VRK1) activates the normally. EMBO J 21, 6225–6235.

2502 FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS S. Blanco et al. Differential stabilization of p53 by VRK2 isoforms

30 Hemann MT, Fridman JS, Zilfou JT, Hernando E, 46 Jackson MW, Agarwal MK, Agarwal ML, Agarwal A, Paddison PJ, Cordon-Cardo C, Hannon GJ & Lowe Stanhope-Baker P, Williams BR & Stark GR (2004) SW (2003) An epi-allelic series of p53 hypomorphs cre- Limited role of N-terminal phosphoserine residues in ated by stable RNAi produces distinct tumor pheno- the activation of transcription by p53. Oncogene 23, types in vivo. Nat Genet 33, 396–400. 4477–4487. 31 Alarcon-Vargas D & Ronai Z (2002) p53-Mdm2 – the 47 Sluss HK, Armata H, Gallant J & Jones SN (2004) affair that never ends. Carcinogenesis 23, 541–547. Phosphorylation of serine 18 regulates distinct p53 func- 32 Woods DB & Vousden KH (2001) Regulation of p53 tions in mice. Mol Cell Biol 24, 976–984. function. Exp Cell Res 264, 56–66. 48 Knights CD, Liu Y, Appella E & Kulesz-Martin M 33 Saito S, Yamaguchi H, Higashimoto Y, Chao C, Xu Y, (2003) Defective p53 post-translational modification Fornace AJ Jr, Appella E & Anderson CW (2003) Phos- required for wild type p53 inactivation in malignant phorylation site interdependence of human p53 post- epithelial cells with mdm2 gene amplification. J Biol translational modifications in response to stress. J Biol Chem 278, 52890–52900. Chem 278, 37536–37544. 49 Sakaguchi K, Saito S, Higashimoto Y, Roy S, Ander- 34 Meek DW (1998) Multisite phosphorylation and the son CW & Appella E (2000) Damage-mediated phos- integration of stress signals at p53. Cell Signal 10, 159– phorylation of human p53 threonine 18 through a 166. cascade mediated by a casein 1-like kinase. Effect on 35 Bode AM & Dong Z (2004) Post-translational modifica- mdm2 binding. J Biol Chem 275, 9278–9283. tion of p53 in tumorigenesis. Nat Rev Cancer 4, 793– 50 Dumaz N, Milne DM & Meek DW (1999) Protein 805. kinase CK1 is a p53-threonine 18 kinase which requires 36 Siliciano JD, Canman CE, Taya Y, Sakaguchi K, prior phosphorylation of serine 15. FEBS Lett 463, Appella E & Kastan MB (1997) DNA damage induces 312–316. phosphorylation of the amino terminus of p53. Genes 51 Stoter M, Bamberger AM, Aslan B, Kurth M, Speidel Dev 11, 3471–3481. D, Loning T, Frank HG, Kaufmann P, Lohler J, 37 de Rozieres S, Maya R, Oren M & Lozano G (2000) Henne-Bruns D et al. (2005) Inhibition of casein kin- The loss of mdm2 induces p53-mediated apoptosis. ase I delta alters mitotic spindle formation and indu- Oncogene 19, 1691–1697. ces apoptosis in trophoblast cells. Oncogene 24, 7964– 38 Ljungman M (2000) Dial 9-1-1 for p53: mechanisms of 7975. p53 activation by cellular stress. Neoplasia 2, 208–225. 52 Chen J, Marechal V & Levine AJ (1993) Mapping of 39 Appella E & Anderson CW (2001) Post-translational the p53 and mdm-2 interaction domains. Mol Cell Biol modifications and activation of p53 by genotoxic stres- 13, 4107–4114. ses. Eur J Biochem 268, 2764–2772. 53 Schon O, Friedler A, Bycroft M, Freund S & Fersht A 40 Brooks CL & Gu W (2003) Ubiquitination, phosphory- (2002) Molecular mechanism of the interaction between lation and acetylation: the molecular basis for p53 regu- MDM2 and p53. J Mol Biol 323, 491–501. lation. Curr Opin Cell Biol 15, 164–171. 54 Jabbur JR, Tabor AD, Cheng X, Wang H, Uesugi M, 41 Ashcroft M, Taya Y & Vousden KH (2000) Stress sig- Lozano G & Zhang W (2002) Mdm-2 binding and TAF nals utilize multiple pathways to stabilize p53. Mol Cell (II) 31 recruitment is regulated by hydrogen bond dis- Biol 20, 3224–3233. ruption between the p53 residues Thr18 and Asp21. 42 Dumaz N, Milne DM, Jardine LJ & Meek DW Oncogene 21, 7100–7113. (2001) Critical roles for the serine 20, but not the ser- 55 Yuan ZM, Huang Y, Ishiko T, Nakada S, Utsugisawa ine 15, phosphorylation site and for the polyproline T, Shioya H, Utsugisawa Y, Yokoyama K, Weichsel- domain in regulating p53 turnover. Biochem J 359, baum R, Shi Y et al. (1999) Role for p300 in stabiliza- 459–464. tion of p53 in the response to DNA damage. J Biol 43 Dumaz N & Meek DW (1999) Serine 15 phosphoryla- Chem 274, 1883–1886. tion stimulates p53 transactivation but does not directly 56 Grossman SR (2001) p300 ⁄ CBP ⁄ p53 interaction and influence interaction with HDM2. EMBO J 18, 7002– regulation of the p53 response. Eur J Biochem 268, 7010. 2773–2778. 44 Lambert PF, Kashanchi F, Radonovich MF, Shiekhat- 57 Ito A, Lai CH, Zhao X, Saito S, Hamilton MH, tar R & Brady JN (1998) Phosphorylation of p53 serine Appella E & Yao TP (2001) p300 ⁄ CBP-mediated p53 15 increases interaction with CBP. J Biol Chem 273, acetylation is commonly induced by p53-activating 33048–33053. agents and inhibited by MDM2. EMBO J 20, 1331– 45 Shieh SY, Ahn J, Tamai K, Taya Y & Prives C (2000) 1340. The human homologs of checkpoint kinases Chk1 and 58 Dornan D, Shimizu H, Perkins ND & Hupp TR (2003) Cds1 (Chk2) phosphorylate p53 at multiple DNA DNA-dependent acetylation of p53 by the transcription damage-inducible sites. Genes Dev 14, 289–300. coactivator p300. J Biol Chem 278, 13431–13441.

FEBS Journal 273 (2006) 2487–2504 ª 2006 The Authors Journal compilation ª 2006 FEBS 2503 Differential stabilization of p53 by VRK2 isoforms S. Blanco et al.

59 Damia G, Filiberti L, Vikhanskaya F, Carrassa L, Taya 66 Kubbutat MHG, Jones SN & Vousden KH (1997) Reg- Y, D’Incalci M & Broggini M (2001) Cisplatinum and ulation of p53 stability by mdm2. Nature 387, 299–303. taxol induce different patterns of p53 phosphorylation. 67 Stewart ZA, Tang LJ & Pietenpol JA (2001) Increased Neoplasia 3, 10–16. p53 phosphorylation after microtubule disruption is 60 Webley K, Bond JA, Jones CJ, Blaydes JP, Craig A, mediated in a microtubule inhibitor- and cell-specific Hupp T & Wynford-Thomas D (2000) Posttranslational manner. Oncogene 20, 113–124. modifications of p53 in replicative senescence overlap- 68 Pospisilova S, Brazda V, Kucharikova K, Luciani MG, ping but distinct from those induced by dna damage. Hupp TR, Skladal P, Palecek E & Vojtesek B (2004) Mol Cell Biol 20, 2803–2808. Activation of the DNA-binding ability of latent p53 61 Milne DM, Palmer RH, Campbell DG & Meek DW protein by protein kinase C is abolished by protein (1992) Phosphorylation of the p53 tumour-suppressor kinase CK2. Biochem J 378, 939–947. protein at the three N-terminal sites by a novel casein 69 Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau kinase I-like . Oncogene 7, 1361–1369. J, Levine AJ & Pavletich NP (1996) Structure of the 62 Milne DM, Campbell DG, Caudwell FB & Meek DW MDM2 oncoprotein bound to the p53 tumor suppressor (1994) Phosphorylation of tumor suppressor protein p53 transactivation domain. Science 274, 948–953. by mitogen-activated protein kinase. J Biol Chem 269, 70 Li M, Luo J, Brooks CL & Gu W (2002) Acetylation of 9253–9260. p53 inhibits its ubiquitination by Mdm2. J Biol Chem 63 Ashcroft M, Kubbutat MH & Vousden KH (1999) Reg- 277, 50607–50611. ulation of p53 function and stability by phosphoryla- 71 Agoulnik AI, Lu B, Zhu Q, Truong C, Ty MT, Arango tion. Mol Cell Biol 19, 1751–1758. N, Chada KK & Bishop CE (2002) A novel gene, Pog, 64 Dornan D & Hupp TR (2001) Inhibition of p53-depen- is necessary for primordial germ cell proliferation in the dent transcription by BOX-I phospho-peptide mimetics mouse and underlies the germ cell deficient mutation, that bind to p300. EMBO Report 2, 139–144. gcd. Hum Mol Genet 11, 3047–3053. 65 Ferguson M, Luciani MG, Finlan L, Rankin EM, 72 Lu B & Bishop CE (2003) Late onset of spermatogen- Ibbotson S, Fersht A & Hupp TR (2004) The develop- esis and gain of fertility in POG-deficient mice indicate ment of a CDK2-docking site peptide that inhibits p53 that POG is not necessary for the proliferation of sper- and sensitizes cells to death. Cell Cycle 3, 80–89. matogonia. Biol Reprod 69, 161–168.

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