The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase
2005; Springer Nature; Volume: 24; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7600584
ISSN1460-2075
AutoresFabien Llambi, Filipe C. Lourenço, Devrim Gözüaçık, Catherine Guix, Laurent Pays, Gabriel del Rio, Adi Kimchi, Patrick Mehlen,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoArticle24 February 2005free access The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase Fabien Llambi Fabien Llambi Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Filipe Calheiros Lourenço Filipe Calheiros Lourenço Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Devrim Gozuacik Devrim Gozuacik Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Catherine Guix Catherine Guix Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Laurent Pays Laurent Pays Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Gabriel Del Rio Gabriel Del Rio The Buck Institute for Age Research, Novato, CA, USA Search for more papers by this author Adi Kimchi Adi Kimchi Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Patrick Mehlen Corresponding Author Patrick Mehlen Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Fabien Llambi Fabien Llambi Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Filipe Calheiros Lourenço Filipe Calheiros Lourenço Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Devrim Gozuacik Devrim Gozuacik Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Catherine Guix Catherine Guix Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Laurent Pays Laurent Pays Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Gabriel Del Rio Gabriel Del Rio The Buck Institute for Age Research, Novato, CA, USA Search for more papers by this author Adi Kimchi Adi Kimchi Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Patrick Mehlen Corresponding Author Patrick Mehlen Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France Search for more papers by this author Author Information Fabien Llambi1, Filipe Calheiros Lourenço1, Devrim Gozuacik2, Catherine Guix1, Laurent Pays1, Gabriel Del Rio3, Adi Kimchi2 and Patrick Mehlen 1 1Apoptosis, Cancer and Development Laboratory, Equipe labellisée 'La Ligue', CNRS FRE2870, Centre Leon Berard, Lyon, France 2Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 3The Buck Institute for Age Research, Novato, CA, USA *Corresponding author. Apoptosis, Cancer and Development Laboratory, CNRS FRE2870, Centre Leon Berard, Lyon, France. Tel.: +33 4 78 78 28 70; Fax: +33 4 78 78 28 87; E-mail: [email protected] The EMBO Journal (2005)24:1192-1201https://doi.org/10.1038/sj.emboj.7600584 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Netrin-1 receptors UNC5H (UNC5H1–4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase. Introduction Netrin-1, a diffusible laminin-related protein, is a bifunctional molecule (Serafini et al, 1994). It plays a major role during nervous system development by mediating chemoattraction and chemorepulsion of axons/neurons by interacting with its main receptors DCC (Deleted in Colorectal Cancer) and UNC5H (Keino-Masu et al, 1996; Serafini et al, 1996; Leonardo et al, 1997). However, netrin-1 was also more recently described as a survival factor. Indeed, both DCC and UNC5H receptors belong to the so-called dependence receptors family (Mehlen et al, 1998; Llambi et al, 2001). Such receptors, which also include p75NTR, RET, Patched, β-integrin and neogenin, share the functional property of inducing cell death when unbound by ligands (for a review, see Mehlen and Bredesen, 2004). Such receptors induce a cellular state of cell dependence toward ligand availability. Thus, DCC or UNC5H expression leads to apoptosis unless netrin-1 is present. While DCC was intensively studied because it was initially proposed as a tumor suppressor (Fearon et al, 1990), the interest in UNC5H receptors emerged only recently (Ackerman et al, 1997; Leonardo et al, 1997). UNC5H receptors, which include four members in mammals (UNC5H1–4), are type I transmembrane receptors. While very little is known about UNC5H4, UNC5H1–3 were shown to induce apoptosis via (i) a cleavage of these receptors by caspase (Llambi et al, 2001; Tanikawa et al, 2003) and (ii) intracellular domains required for cell death, that is, a ZU domain (Williams et al, 2003) and a death domain (Llambi et al, 2001). Interestingly, expression of UNC5H1–3 was shown to be downregulated in various cancers, suggesting that UNC5H receptors may be tumor suppressors that would limit tumor growth by inducing apoptosis outside of ligand availability (Thiebault et al, 2003). Moreover, it was shown that UNC5H2 gene is a direct transcriptional target for the tumor suppressor p53 and mediates, in the conditions tested, p53-induced cell death unless netrin-1 is present (Tanikawa et al, 2003). It is however unclear how UNC5H2 receptor mediates apoptosis. Interestingly, UNC5H proteins display a death domain in their C-terminal extremities related to the death domain of receptors like Fas, TNFr or p75NTR, and the deletion of the death domain of UNC5H2 has been shown to abrogate UNC5H2 proapoptotic activity (Llambi et al, 2001). Sequence analysis reveals that the protein displaying the closest death domain to UNC5H2 death domain is the death-associated protein kinase (DAP-kinase; Deiss et al, 1995; Feinstein et al, 1995) (Figure 1A), that is, 49.4% homology between the two death domains. DAP-kinase is a crucial intracellular protein that mediates cell death induction through a wide spectrum of apoptotic signals via its serine threonine kinase activity (Deiss et al, 1995; Cohen et al, 1999; Jang et al, 2002; Wang et al, 2002). However, the only known signals directly upstream of DAP-kinase that would drive its kinase activity are the calmodulin (CaM) binding through its CaM regulatory region triggered by a calcium spike (Cohen et al, 1997) and an autoinhibitory phosphorylation in this CaM regulatory region, which controls the affinity binding to CaM (Shohat et al, 2001). Because the death domain of DAP-kinase is known to be required for DAP-kinase-induced cell death (Cohen et al, 1999), we investigated the possible link between the transmembrane receptor UNC5H2 that triggers cell death and this cytoplasmic effector for cell death induction. We show here that DAP-kinase is required for a large part of UNC5H2-induced cell death. We also show that the UNC5H2 death domain interacts with the DAP-kinase and regulates the kinase activity of DAP-kinase. We also provide evidence that netrin-1 by interacting with UNC5H2 inhibits DAP-kinase activity, thus suggesting that the dependence receptor UNC5H2 triggers apoptosis by activating DAP-kinase. Figure 1.UNC5H2 interacts with DAP-kinase. (A) Sequence alignment of the death domain of different death domain-containing proteins p75NTR (ngfr), Ankyrin-1 (ank1), DAPK, UNC5H, MyD88 (my88), Fas and RIP. Helices in the death domain of p75NTR (ngfr DD) and of Fas (fas DD) are shown. (B) UNC5H2 interacts with DAP-kinase in HEK 293T. Lysates of HEK 293T transiently transfected with HA-tagged UNC5H1-H2 or H3, together with Flag-tagged DAP-kinase, were subjected to Flag (DAP-kinase) pull-down. Tot: Western blot on lysate before pull-down; IP: α-Flag; WB: α-HA—Western blot with anti-HA antibody after pull-down). (C) Schematic representation of UNC5H2 and the various mutants used in this study. TM: transmembrane domain; DD: death domain; D412: caspase cleavage site. (D) UNC5H2 death domain interacts with DAP-kinase in HEK 293T. Similar experiment as in (B) but HA-tagged DAP-kinase was expressed together with the Flag-tagged UNC5H2-DD. Pull-down here was performed with anti-HA and UNC5H2-DD was analyzed by Western blot using an anti-Flag antibody. (E) UNC5H2 interacts with DAP-kinase through multiple domains in HEK 293T. Similar experiment as in (B) using deletion mutants of UNC5H2. (F) Similar as in (D), but HA-tagged UNC5H2 was expressed together with Flag-tagged DAP-kinase DD and pull-down was performed using anti-FlagM2 antibody. (G) Similar as in (D), but Flag-tagged p75ntr was used instead of UNC5H2. (H) Brains from E13 mice embryos were dissected out and resuspended in the lysis buffer described in Materials and methods supplemented with 1% NP-40 as described before (Forcet et al, 2002). Immunoprecipitation was performed using either a UNC5H2 antibody (α-UNC5H2) or a control antibody (−), and DAP-kinase was detected by immunoblot. Download figure Download PowerPoint Results The death domain of UNC5H2 interacts with DAP-kinase We first analyzed whether UNC5H2 interacts with DAP-kinase. Co-immunoprecipitation experiments were performed from human embryonic kidney (HEK) 293T cells coexpressing Flag-tagged DAP-kinase and HA-tagged UNC5H2. As shown in Figure 1B, HA-tagged UNC5H2 is found immunoprecipitated with Flag-tagged DAP-kinase when anti-FlagM2 antibody was used for pull-down. Interestingly, such interaction can barely be detected when UNC5H3 or UNC5H1, the other members of UNC5H family, is used. To analyze whether the death domain of UNC5H2 is sufficient by itself for the interaction with DAP-kinase, we assessed whether the death domain module of UNC5H2—that is, UNC5H2-DD: UNC5H2 death domain only—was able per se to co-immunoprecipitate with DAP-kinase. As shown in Figure 1D, UNC5H2-DD was pulled down by DAP-kinase when a Flag-tagged UNC5H2-DD-expressing construct was coexpressed in HEK 293T cells together with HA-tagged DAP-kinase-expressing construct and when the immunoprecipitation was performed using anti-HA antibody. To check more precisely whether the death domain of UNC5H2 was the only domain required for interaction with DAP-kinase, we then expressed in HEK 293T cells a series of HA-tagged UNC5H2 mutants truncated at various points within the intracellular domain together with Flag-tagged DAP-kinase. As opposed to our initial expectations, the deletion of the death domain of UNC5H2 was not sufficient to inhibit the interaction between UNC5H2 and DAP-kinase observed after a pull-down performed with anti-FlagM2 antibody (Figure 1E). However, the deletion of the complete intracellular domain of UNC5H2 totally inhibited its interaction with DAP-kinase (Figure 1E). Thus, in addition to the association of DAP-kinase with the death domain of UNC5H2, DAP-kinase indirectly or directly interacts with other domain(s) of UNC5H2. On the other hand, we wondered whether DAP-kinase interacts with UNC5H2 via the death domain. Similarly, while a Flag-tagged DAP-kinase death domain only, that is, DAP-kinase DD, could pull down the HA-tagged UNC5H2 when anti-FlagM2 was used for immunoprecipitation (Figure 1F and not shown), the deletion of the death domain of DAP-kinase was not sufficient to inhibit DAP-kinase/UNC5H2 interactions (data not shown). To discard a nonspecific interaction of DAP-kinase DD with death domain-containing proteins, we assessed whether the closest death domain-containing receptor after UNC5H proteins, that is, p75ntr, interacts with DAP-kinase. HEK 293T cells were then cotransfected with Flag-tagged p75ntr-expressing construct and HA-tagged DAP-kinase-expressing plasmid and HA pull-down was performed. As shown in Figure 1G, p75ntr failed to be pulled down by DAP-kinase, thereby supporting a specific interaction between UNC5H2 and DAP-kinase. Thus, taken together, these data suggest that the UNC5H2/DAP-kinase interaction occurs via their respective death domains and also through other(s) region(s) of both UNC5H2 and DAP-kinase proteins. To assay whether the interaction between DAP-kinase and UNC5H2 may be detected in vivo with endogenous proteins, we performed immunoprecipitations on brains of mice embryo (E13) in which both UNC5H2 and DAP-kinase are known to be expressed (Leonardo et al, 1997; Llambi et al, 2001) (Figure 1H). As shown in Figure 1H, DAP-kinase was pulled down with an antibody raised against UNC5H2, while a nonrelevant antibody, that is, goat polyclonal antibody, was not able to specifically immunoprecipitate DAP-kinase above the low background levels. Thus, either when ectopically expressed in HEK 293T cells or when endogenously expressed in mice embryos, UNC5H2 interacts with DAP-kinase. The UNC5H2 proapoptotic activity is mediated by DAP-kinase We then investigated whether UNC5H2-induced cell death may be mediated by DAP-kinase. In a first attempt to implicate DAP-kinase in UNC5H2-induced cell death, UNC5H2 and DAP-kinase were cotransfected in either HeLa or HEK 293T cells. The presence of DAP-kinase potentiated cell death morphology (i.e., cell detachment, nuclear condensation) in UNC5H2-transfected population, while in the same assay DAP-kinase had no cumulative effect on DCC-induced cell death (Figure 2A and not shown). To further demonstrate the role of DAP-kinase in UNC5H2-mediated cell death, we analyzed whether inhibition of endogenous DAP-kinase activity by dominant-negative mutants may modulate UNC5H2-induced cell death. As previously reported, HEK 293T cells express DAP-kinase endogenously (Cohen et al, 1999). Moreover, while comparing parental HEK 293T and UNC5H2-transfected population, UNC5H2 was detected only in transfected cells (Figure 2B). Along the same lines, the expression levels of UNC5H2 mRNA were estimated by quantitative RT–PCR to be at least 10 times lower than in a normal tissue, for example, in adult human colorectal tissue (not shown), in agreement with the observed loss of UNC5H2 expression in immortalized/transformed cells (Thiebault et al, 2003). In this setting, two different dominant-negative mutants of DAP-kinase, that is, kinase dead mutant (K42A) and the death domain only, were individually coexpressed in HEK 293T cells together with UNC5H2. Cell death was analyzed either by Trypan blue exclusion assay (Figure 2B) or by measurement of caspase activity (not shown). Various plasmid ratios of the UNC5H2-expressing construct and the dominant-negative mutants were used. In most cases, UNC5H2-mediated cell death was inhibited by the presence of these dominant-negative mutants of DAP-kinase (Figure 2B and not shown), while as a negative control, a dominant negative for caspase-8 had no effect (Figure 2B). The 1:2 ratio was found to have the most important inhibitory effect on UNC5H2-induced cell death (Figure 2B). Finally, UNC5H2-induced cell death was analyzed in immortalized murine embryonic fibroblasts (MEFs) derived from either wild-type mouse or DAP-kinase null mouse. UNC5H2- or DCC-expressing construct was transiently transfected in these cells and the UNC5H2- or DCC-transfected cells were concentrated using a microbeads system. While DCC triggered cell death to a similar extent in both parental and DAP-kinase null MEF cells, UNC5H2-induced cell death was reduced in DAP-kinase null MEF cells (Figure 2C). Figure 2.UNC5H2 mediates apoptosis through DAP-kinase. (A) Overexpression of DAP-kinase synergizes UNC5H2-induced cell death. HEK 293T cells were transfected with either UNC5H2- or DCC-expressing construct together or not with DAP-kinase. 48 h after transfection, cell death morphology was examined. Scale bar: 20 μm. (B) Cell death was measured by Trypan blue exclusion assay on HEK 293T cells transfected with UNC5H2 together with dominant-negative mutant of DAP-kinase, that is, the DAP-kinase death domain only, or DAP-kinase K42A, or as a control a dominant-negative mutant of caspase-8. In the assay presented, 0.33 μg of UNC5H2-encoding plasmid versus 0.66 μg of dominant-negative mutant-expressing construct was used for Lipofectamine transfection of 1.5 × 105 cells and the viability of about 900 cells was estimated. Western blots on endogenous UNC5H2 using anti-human UNC5H2 antibody (provided by H Arakawa), HA-tagged UNC5H2 and HA-tagged DAP-kinase mutant using anti-HA antibody were performed. (C) Cell death triggered by DCC or UNC5H2 expression was measured by Trypan blue exclusion assay in MEFs from wild-type mice (+/+) or in MEFs from DAP-kinase knockout mice (−/−). Standard deviations are indicated (n=4). **Student's test, P<0.001. (D) Cell death was analyzed by Trypan blue exclusion assay from HEK 293T cells transfected with DAP-kinase in the presence or not of UNC5H2-DD. Standard deviations are indicated (n=3). Download figure Download PowerPoint It is however interesting to note that neither the dominant-negative mutants nor the use of DAP-kinase null MEF cells completely abrogated the UNC5H2 proapoptotic activity, hence suggesting that besides DAP-kinase, UNC5H2 may also recruit other effector(s) for cell death induction. However, together, these data support the fact that DAP-kinase is involved specifically in UNC5H2-mediated cell death. To investigate whether the UNC5H2 death domain interaction with DAP-kinase was required for this process, we transiently transfected UNC5H2-DD together with DAP-kinase in HEK 293T cells. The death domain of UNC5H2 was not able to trigger cell death by itself. The expression of DAP-kinase alone triggered cell death to a limited extent (37%), while coexpression of DAP-kinase and the UNC5H2 death domain significantly enhanced 293T cell death (52%) (Figure 2D). Thus, UNC5H2 interacts with DAP-kinase and requires, at least in part, DAP-kinase to behave as a proapoptotic receptor probably because of the interaction between UNC5H2 death domain and DAP-kinase. UNC5H2 triggers the DAP-kinase catalytic activity In light of previous studies documenting that DAP-kinase mediates apoptosis through its catalytic activity (Cohen et al, 1997), we further investigated whether its interaction with UNC5H2 may lead to activation of the kinase catalytic activity. DAP-kinase activity has been shown to be tightly controlled by an inhibitory type of autophosphorylation. As previously shown, in the absence of a calcium spike, DAP-kinase is autophosphorylated on Ser308 located in the CaM-binding domain, a process that turns off the kinase activity (Cohen et al, 1997; Shohat et al, 2001). Monoclonal antibodies directed against phospho-Ser308 or immunoprecipitation with a phosphoserine antibody detected this phosphorylation event. HEK 293T cells were then transiently transfected with DAP-kinase- and/or HA-tagged UNC5H2-expressing constructs in the presence or absence of netrin-1. In a first assay, autophosphorylation of DAP-kinase was analyzed by performing an immunoprecipitation with a phosphoserine antibody followed by a DAP-kinase immunoblot (Figure 3A). In a second assay, an immunoblot was directly performed using a monoclonal antibody directed against phospho-Ser308 of DAP-kinase (Figure 3B). In a third assay, this phospho-antibody was used to perform immunostaining (Figure 3C and D). In both cases, it was observed that the expression of UNC5H2 in HEK 293T cells leads to the inhibition of DAP-kinase autophosphorylation on Ser308 (Figure 3A–D). Interestingly, when UNC5H2 is expressed, the presence of netrin-1 completely restored DAP-kinase autophosphorylation. Thus, the pair UNC5H2/netrin-1 regulates autophosphorylation of DAP-kinase. Figure 3.The pair netrin-1/UNC5H2 modulates DAP-kinase activity. (A) DAP-kinase phosphorylation is regulated by the pair UNC5H2/netrin-1. HEK 293T cells were transiently transfected with a control vector or with HA-tagged UNC5H2-expressing construct together with DAP-kinase-expressing construct. Netrin-1 was added 24 h after transfection. Pull-down with a phospho-serine antibody followed by Western blot using anti-DAP-kinase was performed. (B) Similar transfection assay and netrin-1 incubation as in (A) but a series of Western blot was performed using either anti-DAP-kinase, anti-HA (UNC5H2) or anti-phospho-DAP-kinase. (C) Phospho-DAP-kinase immunostaining on HEK 293T transfected with HA-tagged UNC5H2-expressing construct together with DAP-kinase-expressing construct. Representative images are shown. DAPI staining was performed to monitor the total number of cells. Scale bar: 20 μm. (D) Quantitative analysis from (C) was performed by counting the number of cells stained by the phospho-DAP-kinase antibody. About 1000 total cells were counted in an average of 50 fields from five different experiments. (E) DAP-kinase activity is regulated by the pair UNC5H2/netrin-1. Lysates from HEK 293T transfected with Flag-tagged DAP-kinase together with UNC5H2 or Bax, incubated or not with netrin-1 (0.3 μg/ml, 24 h), were subjected to anti-Flag (DAP-kinase) pull-down and assessed for DAP-kinase phosphorylating activity in vitro as described in Materials and methods. To induce calcium-mediated DAP-kinase activation, CaM (1 μM)/Ca2+ (0.5 mM) was added to the kinase assay reaction medium during 10 min. Standard deviations are indicated (n=5). **Student's test, P<0.005. Insets: Control Western blots showing DAP-kinase expression level (α-Flag) and DAP-kinase autophosphorylation (α-P-DAPK). (F) Similar experiment as in (E) but HEK 293T cells were cultured in Ca2+-free medium 24 h after transfection. (G) Semidissociated P5 rat hippocampi were incubated for 1 h with purified netrin and subjected to lysis and DAP-kinase pull-down using the anti-DAP-kinase antibody described in Shani et al (2004). DAP-kinase catalytic activity was then measured as in (E). Standard deviations are indicated (n=4). Inset: Control Western blot showing DAP-kinase expression level. Download figure Download PowerPoint To demonstrate that the regulation of autophosphorylation by this pair consequently affects DAP-kinase activity, we performed in vitro kinase assays using the DAP-kinase substrate peptide KKRPQRRYSNVF. HEK 293T cells were then transfected with Flag-tagged DAP-kinase and/or UNC5H2-expressing constructs in the presence or absence of netrin-1. Immunoprecipitations were then performed using anti-FlagM2 antibody and the resulting pull-downs were tested for their ability to phosphorylate in vitro the DAP-kinase substrate. It was found that while UNC5H2 increases DAP-kinase activity to a similar extent as a treatment with CaM/calcium, the presence of netrin-1 is sufficient to block this effect (Figure 3E). To further confirm that these general changes observed in DAP-kinase activity were related to a specific effect of UNC5H2, bound or unbound to netrin-1, rather than to an effect of UNC5H2 on cell death that consequently affects DAP-kinase activity, we assessed whether Bax, a general apoptosis inducer, also triggered DAP-kinase activation. As shown in Figure 3E, Bax transfection while affecting caspase activity (data not shown) had no effect on DAP-kinase activity. Thus, the pair UNC5H2/netrin-1 regulates DAP-kinase activity. Moreover, the UNC5H2 death domain that was previously shown to interact with DAP-kinase is required for this activation since transfection of a mutant of UNC5H2 deleted in its death domain instead of full-length UNC5H2 impaired DAP-kinase catalytic activity. To further confirm the role of the UNC5H2 death domain, HEK 293T cells were transfected with UNC5H2-DD instead of full-length UNC5H2. This resulted in a significant increase in DAP-kinase activity, demonstrating that the death domain of UNC5H2 per se activates DAP-kinase. Because of the calcium-dependent DAP-kinase activity (Cohen et al, 1997), we next wondered whether UNC5H2 modulates DAP-kinase activity through calcium modulation. Similar DAP-kinase catalytic activity assays were then performed from transfected HEK 293T cells cultured in medium devoid of calcium/magnesium. As shown in Figure 3F, no change in UNC5H2-mediated DAP-kinase catalytic activity was observed, hence supporting a direct effect of UNC5H2-DD on DAP-kinase activity. To further analyze in vivo relevance of this UNC5H2-mediated DAP-kinase activation, we next explored whether in vivo netrin-1 level may affect DAP-kinase catalytic activity. The hippocampus or cerebellum of P5 rat was dissected out, semidissociated and further incubated or not with netrin-1. To first determine whether DAP-kinase catalytic activity can be detected in the hippocampus, pull-downs were performed using either an anti-DAP-kinase antibody or an unrelated antibody, that is, goat polyclonal antibody, and the DAP-kinase catalytic activity within the pull-downs was assessed by measuring KKRPQRRYSNVF phosphorylation. Specific DAP-kinase catalytic activity can be detected in the hippocampus (Figure 3G). A similar experiment was then performed after incubation of semidissociated hippocampus with netrin-1. As shown in Figure 3G, DAP-kinase catalytic activity was significantly reduced, hence strengthening the in vivo relevance of the UNC5H2/DAP-kinase interaction. Interestingly, when a similar experiment was performed in semidissociated cerebellum, DAP-kinase catalytic activity was not modulated by the presence of netrin-1 (not shown), hence suggesting that the effect observed in the hippocampus is a tissue-specific effect. UNC5H2 cleavage is a prerequisite for its death signaling through DAP-kinase One common feature of dependence receptors is the fact that most of them have been shown to be cleaved by caspase, a cleavage required, at least in part, for cell death induction by these receptors (Mehlen and Bredesen, 2004). Hence, UNC5H1–3 were shown to be cleaved in vitro, and mutation of the caspase cleavage site of UNC5H2 reduced or turned off UNC5H2 proapoptotic activity (Llambi et al, 2001; Tanikawa et al, 2003). We then investigated whether DAP-kinase autophosphorylation and activity were dependent on UNC5H2 cleavage by measuring DAP-kinase Ser308 phosphorylation and catalytic activity in the presence of UNC5H2 D412N, a mutant of UNC5H2 unable to be cleaved (Llambi et al, 2001), or in the presence of a mutant of UNC5H2 lacking the whole C-terminal domain lying below the caspase cleavage site. HEK 293T cells were then transiently transfected with either wild-type UNC5H2-, UNC5H2 D412N- or UNC5H2 1–412-expressing construct. Autophosphorylation was then monitored by immunoblot using the phospho-specific antibody directed against DAP-kinase Ser308, while DAP-kinase catalytic activity was determined by measuring the ability of DAP-kinase pull-down to phosphorylate the DAP-kinase substrate peptide. As shown in Figure 4, the D412N mutant form was less efficient than the wild-type UNC5H2 in reducing DAP-kinase autophosphorylation (Figure 4A) and in inducing the DAP-kinase phosphorylating activity (Figure 4B). The mutant of UNC5H2 lacking the cleaved fragment was unable to either inhibit DAP-kinase autophosphorylation or to enhance DAP-kinase catalytic activity (Figure 4B and data not shown). It is also interesting to note that the amplitude of reduction in DAP-kinase activity from wild-type UNC5H2 to UNC5H2 D412N was comparable to the reduction of the UNC5H2 proapoptotic activity observed after D412N mutation (Llambi et al, 2001). Thus, UNC5H2-mediated DAP-kinase activation probably requires the preliminary caspase cleavage of UNC5H2. Figure 4.DAP-kinase autophosphorylation and activation by UNC5H2 is dependent on UNC5H2 caspase cleavage. (A) Similar experiment as in Figure 3A but HEK 293T cells were transfected with either full-length UNC5H2 or UNC5H2 mutated in the caspase site (D412N). (B) Similar experiment as in Figure 3E but UNC5H2 D412N or UNC5H2 1–412, that is, UNC5H2 deleted below the caspase cleavage site, was used. Standard deviations are indicated (n=5). **Student's test, P<0.005. Inset: Control Western blot showing expression of UNC5H2 and its related mutants. Download figure Download PowerPoint Discussion Dependence receptors can be viewed as membrane sensors for cell fate decision. In the pres
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