Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase. Effect on Mdm2 binding

The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).     

Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.     

Roles of VRK1 as a new player in the control of biological processes required for cell division

Cell division, in addition to an accurate transmission of genetic information to daughter cells, also requires the temporal and spatial coordination of several biological processes without which cell division would not be feasible. These processes include the temporal coordination of DNA replication and chromosome segregation, regulation of nuclear envelope disassembly and assembly, chromatin condensation and Golgi fragmentation for its redistribution into daughter cells, among others. However, little is known regarding regulatory proteins and signalling pathways that might participate in the coordination of all these different biological functions. Such regulatory players should directly have a role in the processes leading to cell division. VRK1 (Vaccinia-related kinase 1) is an early response gene required for cyclin D1 expression, regulates p53 by a specific Thr18 phosphorylation, controls chromatin condensation by histone phosphorylation, nuclear envelope assembly by phosphorylation of BANF1, and participates in signalling required for Golgi fragmentation late in the G2 phase. We propose that VRK1, a Ser-Thr kinase, might be a candidate to play an important coordinator role in these cell division processes as part of a novel signalling pathway.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

VRK1 interacts with p53 forming a basal complex that is activated by UV-induced DNA damage

DNA damage immediate cellular response requires the activation of p53 by kinases. We found that p53 forms a basal stable complex with VRK1, a Ser–Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. We propose that the VRK1–p53 basal complex is an early-warning system for immediate cellular responses to DNA damage.     

Kinetic properties of p53 phosphorylation by the human vaccinia-related kinase 1

The vaccinia-related kinase 1 (VRK1) protein is a nuclear Ser-Thr kinase that phosphorylates p53 in Thr18. We have determined the enzyme properties regarding its different substrates. VRK1 has a high affinity for ATP (K(m) 50 microM) and is thus saturated by the intracellular concentration of ATP in vivo. VRK1 uses preferentially magnesium, but is also functional with manganese and zinc. The VRK1 protein is autophosphorylated in multiple residues without effect on its activity. One autophosphorylated residue, T355, is within the VRK1 regulatory carboxy terminus. The kinase phosphorylates p53 with a K(m) of 1 microM and is well suited to respond to the variations of intracellular p53 concentration, which fluctuates as a response to different types of cellular stress.     

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.     

Nuclear extracellular signal-regulated kinase 2 phosphorylates p53 at Thr55 in response to doxorubicin.

In this study, we showed that nuclear ERK2 phosphorylates p53 at Thr55 in response to doxorubicin. p53 was found to physically interact with ERK2 as evidenced by Western blotting of ERK2 coimmunoprecipitated complex. The gene fragment encoded for N-terminal 68 amino acids was subcloned and fused with 6-His. Each serine or threonine site in this fragment, the possible phosphorylation site, was mutated to alanine. The recombinant proteins were used as substrates in ERK2 kinase assay. The results show that ERK2 phosphorylated p53 at Thr55. Further, electromobility shift assay showed that the phosphorylation of p53 by nuclear ERK2 was closely related to the transactivating activity of p53. These findings suggest that ERK2 may play a role in response to DNA damage via interaction with p53.     

Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b (RSK-2)

Regulatory light chain of myosin II (MRLC) was identified as a novel substrate of p90 ribosomal S6 kinase (RSK)-2, a Ser/Thr protein kinase which is phosphorylated and activated by mitogen-activated protein kinase (MAPK) in vitro and in vivo. Phosphopeptide map of MRLC phosphorylated by RSK-2 was identical to that by myosin light chain kinase (MLCK). Phosphoserine was recovered by the phosphoamino acid analysis of MRLC phosphorylated by RSK-2. Further, phosphorylation using recombinant glutathione S-transferase (GST) fusion proteins of HeLa MRLC2 revealed that RSK-2 phosphorylated wild-type MRLC2 (GST-wtMRLC2) but not its mutants GST-MRLC2(S19A) or GST-MRLC2(T18AS19A) (alanine substituted for Ser19 or both Ser19 and Thr18). These results revealed that RSK-2 phosphorylates MRLC at Ser19 as did MLCK. Phosphorylation of myosin II by RSK-2 resulted in activation of actin-activated MgATPase activity of myosin II. Interestingly, RSK-2 activity to phosphorylate MRLC was suppressed by phosphorylation with MAPK. RSK-2 might be a mediator that regulates myosin II activity through the MAPK cascade.     

Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage-induced apoptosis

Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage-induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner.     

Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.     

Sensitivity of the kinase activity of human vaccinia-related kinase proteins to toxic metals

The human vaccinia-related kinase (VRK) proteins VRK1 and VRK2 regulate different processes, such as the cell cycle, DNA damage response, and signaling by mitogen-activated protein kinases in response to growth factors or cellular stress. Alterations in expression levels of these Ser–Thr kinases are associated with cancer and neurodegenerative diseases. These functions suggest that they might also be targets of toxic metals, and thus contribute to the pathogenic effects associated with metal intoxication. VRK1 is inhibited by cadmium, copper, and mercury, and VRK2 is more sensitive to cadmium and much less sensitive to copper and mercury. Both kinases are insensitive to lead and cobalt. VRK1 is in general more sensitive than VRK2 in the low micromolar range. This inhibitory effect induced by these metals was detected in an autophosphorylation assay, as well as in phosphorylation assays using p53 and histone H3 as substrates. The accumulation of these three metals in cells can contribute, by inhibition of VRKs, to their toxic pathogenic effects, particularly their neurological manifestations. In this context copper has not generally been associated with any intoxication syndrome, except Wilson’s syndrome, but it might be implicated in some alterations with which it has not yet been associated.     

Differential inhibitor sensitivity between human kinases VRK1 and VRK2

Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 μM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31-8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer.