Par-4 is an essential downstream target of DAP-like kinase (Dlk) in Dlk/Par-4-mediated apoptosis.

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A-expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.

Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants.

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability.

Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation.

Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.

Characterization of rat BLOS2/Ceap, a putative yeast She3 homolog, as interaction partner of apoptosis antagonizing transcription factor/Che-1.

AATF/Che-1 is a coactivator of several transcription factors, including steroid hormone receptors. In search of novel interaction partners of AATF, we identified BLOS2 (BLOC1S2, also termed Ceap) from a rat cDNA library. BLOS2 is extremely conserved with a high degree of homology to yeast She3p. The clone isolated represents a splice variant encoding a polypeptide of 168 residues. Rat BLOS2 mRNA is highly expressed in brain and testis and at lower levels in other tissues, but not in skeletal or smooth muscle. Expression as a tagged fusion protein revealed predominant cytoplasmic, but also nuclear localization. In the cytoplasm, BLOS2 fusion proteins exhibit diffuse, filamentous, or dotted distribution, with the latter partially co-localizing with recycling endosomes. In addition, BLOS2 localizes to centrosomes or the pericentrosomal region. Moreover, BLOS2 co-localizes with myosin V globular tail domains in vesicle-like structures. However, a direct interaction could not be demonstrated. In transactivation assays, BLOS2 enhanced transcription from androgen receptor and p53-responsive promoters. However, this enhancement correlated with accumulation of both androgen receptor and p53, suggesting that BLOS2 has a stabilizing effect on these transcription factors. We propose that BLOS2 functions as an adapter in processes such as protein and vesicle processing and transport, and perhaps transcription.

Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis.

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis.

Ectopic expression of Par-4 leads to induction of apoptosis in CNS tumor cell lines.

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein originally identified as a gene product upregulated in prostate tumor cells undergoing apoptosis. Down-regulation of Par-4 has been linked to several cancers. Since Par-4 also plays a crucial role in neuronal apoptosis, we investigated the expression of Par-4 in tumor cell lines derived from representative tumor types of the CNS, including primitive neuroectodermal tumor (PNET), medulloblastoma, neuroblastoma and glioma of human, rat and murine origin. We show that Par-4 is frequently down-regulated, either transcriptionally or post-transcriptionally in the CNS tumor cell lines. Moreover, we demonstrate that ectopic expression of Par-4 is sufficient to directly induce apoptosis in these CNS tumor cells, in contrast to other cancer cells where replenishment of Par-4 levels only sensitizes the cells to apoptotic stimuli. Induction of apoptosis by Par-4 in the neural tumor cell lines is independent of endogenous Bcl-2 levels and PKCzeta activity, although it has been proposed that Par-4 can exert its pro-apoptotic function by down-modulation of Bcl2 expression and inhibition of PKCzeta. Co-expression of Par-4 and a dominant-negative mutant of FADD resulted in a slight reduction of apoptosis in some tumor cell lines, indicating that Par-4 may partially induce apoptosis via the Fas death pathway. Furthermore, these data suggested that the pro-apoptotic function of Par-4 involves (an)other yet unidentified apoptotic pathway(s) in the CNS tumor cell lines. Since Par-4 by itself is not sufficient to induce apoptosis in non-tumor cells, reintroduction of Par-4 into primary CNS tumors or reactivation of the pathways of Par-4-mediated apoptosis represent promising targets in anti-tumor therapy.

TSG101 interacts with apoptosis-antagonizing transcription factor and enhances androgen receptor-mediated transcription by promoting its monoubiquitination.

Apoptosis-antagonizing transcription factor (AATF), also termed Che-1, was identified as interacting protein of Dlk/ZIP kinase and RNA polymerase II, respectively. Che-1 has additionally been shown to bind Rb, thereby activating transcription factor E2F and promoting cell cycle progression. Moreover, AATF enhances steroid receptor-mediated transactivation in a hormone- and dose-dependent manner (Leister, P., Burgdorf, S., and Scheidtmann, K. H., (2003) Signal Transduction 3, 18-25). These data suggest that AATF exerts its functions through interaction with different transcription factors. In search of novel interaction partners of AATF, we identified the tumor susceptibility gene product TSG101, which had also been recognized as a co-regulator of nuclear hormone receptors. Interestingly, TSG101 and AATF functioned as cooperative coactivators in androgen receptor-mediated transcription. Because TSG101 was also shown to play a role in regulation of ubiquitin conjugation, we asked whether its coactivating function might be linked to ubiquitination. Indeed, TSG101 enhanced monoubiquitination of the androgen receptor in a ligand-dependent manner, and this correlated with enhanced transactivating capacity. Furthermore, a dominant-negative mutant of ubiquitin preventing polyubiquitination also stimulated androgen receptor-mediated transcription, which in this case could not be enhanced by TSG101. We propose that TSG101 activates androgen receptor-induced transcription by transient stabilization of the monoubiquitinated state, thus revealing a novel regulatory mechanism for nuclear receptors.

DAP-like kinase, a member of the death-associated protein kinase family, associates with centrosomes, centromers, and the contractile ring during mitosis.

DAP-like kinase (Dlk) is a nuclear serine/threonine-specific kinase which has been implicated in apoptosis. However, induction of apoptosis by Dlk requires its relocation to the cytoplasm, particularly association with the actin cytoskeleton, which is achieved through interaction with pro-apoptotic protein Par-4. On the other hand, nuclear Dlk does not induce apoptosis and has rather been implicated in transcription. To further explore the biological functions of Dlk, we established a cell clone of MCF-7 cells stably expressing a GFP-Dlk fusion protein at low level. Ectopic expression of GFP-Dlk did not affect the growth properties of the cells. During interphase, GFP-Dlk showed a diffuse nuclear distribution with punctate staining in a subpopulation of cells. During mitosis, however, Dlk was associated with centrosomes, centromeres, and the contractile ring, but not with the mitotic spindle. Association with centrosomes, as confirmed by colocalization with gamma-tubulin and pericentrin persisted throughout mitosis but was also seen in interphase cells. Interestingly, GFP-Dlk and gamma-tubulin could be co-immunoprecipitated indicating that they are present in the same protein complex. Association of Dlk with centromeres, as verified by confocal fluorescence microscopy with centromere-specific antibodies was more restricted and discernable from prophase to early anaphase. Centromere association of Dlk coincides with H3 phosphorylation at Thr11 that is specifically phosphorylated by Dlk in vitro (U. Preuss, G. Landsberg, K. H. Scheidtmann, Nucleic Acids Res. 31, 878-885, 2003). During cytokinesis, Dlk was enriched in the contractile acto-myosin ring and colocalized with Ser19-phosphorylated myosin light chain, which is an in vitro substrate of Dlk. Strikingly, a C-terminal truncation mutant of Dlk generated multi-nucleated cells. Together, these data suggest that Dlk participates in regulation and, perhaps, coordination of mitotis and cytokinesis.

Novel mitosis-specific phosphorylation of histone H3 at Thr11 mediated by Dlk/ZIP kinase.

Death-associated protein (DAP)-like kinase (Dlk), also known as Zipper interacting protein (ZIP) kinase, is a nuclear serine/threonine-specific kinase that phosphorylates core histones H3 and H4, and myosine light chain in vitro. It interacts with transcription and splicing factors as well as with pro-apoptotic protein Par-4 suggesting that it participates in multiple cellular processes. To explore the significance of histone phosphorylation by Dlk, we determined the phosphorylation site in H3 and generated phosphospecific antibodies for in vivo analyses. Interestingly, Dlk/ZIP kinase phosphorylated histone H3 at a novel site, Thr11, rather than Ser10, which is characteristic of mitotic chromosomes. Immunoblotting and confocal immunofluorescence analyses demonstrated that phosphorylation of H3 at Thr11 occurred in vivo and was restricted to mitosis as well. It was discernable from prophase to early anaphase and particularly enriched at centromeres. Strikingly, during this time interval, Dlk was associated with centromeres too, as revealed by stable expression of a green fluorescent protein (GFP)-Dlk fusion protein. These findings strongly suggest that Dlk is a centromere-specific histone kinase that might play a role in labeling centromere-specific chromatin for subsequent mitotic processes.

DNA substrate dependence of p53-mediated regulation of double-strand break repair.

DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.

DAP-like kinase interacts with the rat homolog of Schizosaccharomyces pombe CDC5 protein, a factor involved in pre-mRNA splicing and required for G2/M phase transition.

DAP-like kinase (Dlk, also termed ZIP kinase) is a leucine zipper-containing serine/threonine-specific protein kinase with as yet unknown biological function(s). Interaction partners so far identified are either transcription factors or proteins that can support or counteract apoptosis. Thus, Dlk might be involved in regulating transcription or, more generally, survival or apoptosis. Here we report on a new interaction partner, the rat homolog of Schizosaccharomyces pombe CDC5 protein, a presumptive transcription and splicing factor involved in the G(2)/M transition. In vitro, rat CDC5 forms complexes with, but is not phosphorylated by, Dlk. Rather, it was phosphorylated by an associated kinase which was identified as CK2. The interaction domain of Dlk was mapped to the leucine zipper, while that of CDC5 was mapped to the C-terminal region between residues 500 and 802. In vivo, both proteins co-localize perfectly in distinct speckle-like structures in the nucleus, some of which overlap with promyelocytic leukemia protein. Interestingly, splicing factor SC35, which also resides in speckles, was partially displaced upon overexpression of either CDC5 or Dlk, perhaps due to phosphorylation by Dlk. Together with previous data, these results suggest that Dlk might play a role in coordinating specific transcription and splicing events.

The rat homologue of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) associates with actin filaments, recruits N-WASP from the nucleus, and mediates mobilization of actin from stress fibers in favor of filopodia formation.

We cloned and characterized the rat homologue of the Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Rat WIP shows 86% amino acid sequence identity to human WIP. Northern analyses revealed two major mRNA species of 5.0 and 3.8 kb, which were ubiquitously expressed, though predominantly in spleen and lung. Minor species of 2.4, 1.8, 1.4, and 1.1 kb were also detected in some tissues and cell lines. Thus, WIP is subject to tissue-specific alternative splicing. WIP bound to N-WASP in vivo, as revealed by co-immunoprecipitation. Expression of WIP in rat fibroblasts revealed a clear co-localization with actin stress fibers. However, expression in tumor cells lacking actin cables did not restore these structures. Interestingly, co-expression of WIP and N-WASP resulted in redistribution of N-WASP, abrogating its dominant nuclear expression and leading to co-localization with WIP in the perinuclear area and with actin in membrane protrusions. Moreover, stress fibers and, concomitantly, the associated WIP were largely dissolved. Very similar effects were seen upon epidermal growth factor stimulation of serum-starved cells. Our results suggest that WIP might be involved in transmitting mitogenic signals to cytoskeletal functions, perhaps by modulating the subcellular localization of N-WASP. Interaction of N-WASP with WIP may in turn lead to mobilization of actin from stress fibers and nucleation of new actin filaments in filopodia.