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Physical and functional interaction between BH3 J Sunayama1,2,3, Y Ando2, N Itoh2, A Tomiyama2, K Sakurada2, A Sugiyama3, D Kang4, F Tashiro3, Y Gotoh1, Y Kuchino2,5,6 and C Kitanaka2,7Received 12 November 2003; Revised 28 January 2004; Accepted 4 February 2004; Published online 19 March 2004.

Top of pageAbstractBcl 2 homology domain (BH) 3 only proteins of the proapoptotic Bcl 2 subfamily play a key role as initiators of mitochondria dependent apoptosis. To date, at least 10 mammalian BH3 only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic leather pandora bracelet signals to mitochondria. Hrk/DP5 is one of the mammalian BH3 only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk mediated apoptosis, we have conducted yeast two hybrid screening for Hrk interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C terminal region of p32. Importantly, Hrk induced apoptosis was suppressed by the expression of p32 mutants lacking the N terminal mitochondrial signal sequence (p32(74 and the conserved C terminal region (p32 (1 which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA mediated knockdown of p32 conferred protection against Hrk induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk mediated apoptosis. Keywords: Hrk, DP5, p32, mitochondria, Bcl 2, BH3 onlyTop of pageIntroductionThe Bcl 2 protein family, composed of members that promote (proapoptotic) and inhibit (antiapoptotic) apoptosis, plays a central role in apoptosis regulation through the modulation of the permeability of the mitochondrial membrane against apoptogenic molecules such as cytochrome c and Smac/DIABLO.1, 2, 3, 4 The proapoptotic members of the Bcl 2 family are further categorized into two subgroups, those having Bcl 2 homology (BH) domains 1 ('multidomain' members, represented by Bax and Bak) and those having only the BH3 domain ('BH3 only' members, represented by Bid, Bad, Bik, and Hrk).4 Accumulating evidence suggests that the BH3 only members and the multidomain members have distinct roles in apoptosis regulation, acting, respectively, as initiators and downstream signal transducers of apoptosis: upon receiving apoptotic stimuli, the BH3 only members are activated primarily via transcriptional and post translational mechanisms, which then activate the downstream multidomain members to permeabilize the mitochondrial membrane.4, 5, 6, 7 In contrast to the case of Caenorrhabditis elegans, whereby the only BH3 protein EGL 1 serves as the initiator for all developmental apoptosis of somatic cells, mammals have at least 10 BH3 only proteins that differ in their expression pattern and mode of activation, which may imply that, in mammals, each BH3 only protein functions as a stimuli or cell type specific initiator of apoptosis.8 Hrk/DP5 is one of the mammalian BH3 only proteins identified during screening for proteins that interact with Bcl 2 or are upregulated in nerve growth factor (NGF) deprivation induced death of sympathetic neuron.9, 10 The initial studies demonstrated that Hrk expression is relatively restricted to the brain and in the lymphoid tissues,9, 10 suggesting that Hrk may have a specific role in these tissues. Consistent with this idea, induction of Hrk expression has been demonstrated in NGF deprived sympathetic neurons,9 in apoptosis of cortical neurons after amyloid protein exposure,11 in apoptotic cerebellar granule neurons cultured under a low potassium condition,12 in spinal neurons of amyotrophic lateral sclerosis patients,13 in retinal ganglion cells of axontomized retina,14 as well as in growth factor deprived hematopoietic cells.15 Although Hrk expression has also been detected in blastomere undergoing fragmentation during embryonic development,16 these characteristic patterns of Hrk expression strongly suggest that this BH3 only protein may have a specific role in initiating neuronal and hematopoietic cell apoptosis under physiological and pathological conditions. Despite such potential significance of Hrk in physiological and/or pathological apoptosis, the mechanism of Hrk mediated apoptosis has been poorly investigated. Although recent reports indicated that transcription of the hrk gene is under the control of the c Jun N terminal kinase pathway12 as well as a transcriptional repressor DREAM17 and that Hrk induced apoptosis requires the expression of Bax,12 the molecular mechanisms involved in the regulation of Hrk mediated apoptosis still remain largely obscure. In this study, in an attempt to identify cellular proteins that are involved in the regulation of Hrk mediated apoptosis, we carried out yeast two hybrid screening and isolated p32 as a novel, Hrk interacting protein. p32 is a homotrimeric protein localized predominantly in mitochondria, whose physiological function in mammalian mitochondria remains unknown.18, 19 Functional analyses of Hrk and p32 revealed that Hrk is targeted to mitochondria to interact with p32 and that interaction with a functional p32 trimer is essential for Hrk to exert its proapoptotic activity. Thus, our data suggest a critical role for p32 in Hrk mediated apoptosis. Top of pageResultsIsolation of p32 as an Hrk interacting cellular factor by yeast two hybrid screeningTo identify cellular proteins that interact with Hrk, we conducted yeast two hybrid screening of a human placenta cDNA library using Hrk as a bait. Screening of 6.4 106 cDNA clones yielded 24 positives, 12 of which proved to activate the reporter genes in an Hrk dependent manner (true positives). One of the 12 true positives encoded Bcl xL, an antiapoptotic Bcl 2 family member previously shown to bind Hrk,10 and four other clones encoded Mcl 1, another antiapoptotic member expected to interact with Hrk similarly to Bcl xL. These results underscored the idea that antiapoptotic members are interacting partners for BH3 only members,20 and also suggested that the screening system worked properly. Interestingly, one of the remaining clones contained the entire coding region for p32,18, 21 in frame to the GAL4 activation domain (GAL4AD). The specificity of the interaction between p32 and Hrk in yeast cells was further confirmed by reintroducing the GAL4AD p32 expression vector (pACT2 p32) into yeast cells together with expression vectors producing GAL4 DNA binding domain (GAL4DBD) alone (pGBT9) or GAL4DBD fused to irrelevant proteins such as p53 (pVA3) and lamin C (pLAM5') (Figure 1). p32 is a mitochondrial protein whose physiological function remains unknown, but recent crystal structure analysis suggested a possible role for p32 in apoptosis regulation.19 We therefore characterized p32 further in this study as a possible cellular factor that interacts with Hrk both physically and functionally. Yeast cells were transformed with the pACT2 p32 plasmid encoding p32 fused to the GAL4 activation domain together with a plasmid encoding either the GAL4DBD alone (pGBT9) or GAL4DBD fused to FLAG Hrk (pGBT9 FLAG Hrk), mouse p53 (pVA3), or human lamin C (pLAM5'). Growth of yeast in the absence of histidine and positive X Gal staining is indicative of protein interaction Full figure and legend (321K) Binding of Hrk and p32 in vitro and in mammalian cellsWe further tested the specific interaction pandora charms stores between Hrk and p32 observed in the yeast two hybrid system through independent approaches. First, the entire coding region of p32 cDNA was transcribed and translated in vitro, and the in vitro translation product was tested for its ability to bind to the Hrk protein fused to glutathione S transferase (GST). The full length p32 protein efficiently bound to GST FLAG Hrk but not to GST alone, suggesting that p32 binds Hrk in vitro (Figure 2a). However, p32 did not bind to any of these proteins under the assay condition, underscoring the specificity of the binding (Figure 2a). We next examined whether Hrk expressed in mammalian cells could bind to p32 fused to GST. For this, FLAG tagged Hrk was expressed in COS cells, and the cell lysates were incubated with GST alone or with GST fused to Bcl xL or to full length p32. As shown in Figure 2b, GST HA p32 and GST Bcl xL, but not GST alone, pulled down comparable amounts of FLAG Hrk, suggesting that p32 binds Hrk as efficiently as Bcl xL. Thus, the results of the in vitro binding analyses provided clear evidence that Hrk binds p32 in a specific manner. We then asked whether Hrk interacts with p32 in mammalian cell lysates. COS cell lysates expressing both FLAG Hrk and the full length p32 tagged with an Myc epitope at the C terminus cheap pandora beads (p32(1 were subjected to immunoprecipitation by an anti FLAG antibody as well as by a control antibody, and the immunoprecipitates were analyzed by immunoblotting with an anti Myc tag antibody. As shown in Figure 2c, p32 Myc was co precipitated with FLAG Hrk by the anti FLAG antibody but not by the control antibody. We further wished to determine whether FLAG Hrk interacts with endogenous, not exogenously overexpressed, p32 in the course of Hrk induced apoptosis. To this end, lysates were prepared from COS cells transiently transfected with FLAG Hrk, immunoprecipitated (IP) with the anti FLAG and the control antibodies, and the presence of endogenous p32 in the immunoprecipitates was probed by anti p32 antibody. The results clearly indicated that endogenous p32 is co precipitated specifically with FLAG Hrk (Figure 2d), suggesting that Hrk binds endogenous p32 during Hrk induced apoptosis. (a) [35S] labeled p32 protein produced by in vitro transcription and translation was pulled down with the indicated GST fusion proteins and detected by autoradiography after SDS PAGE. (b) COS 7 cell lysates transiently transfected with a plasmid expressing FLAG Hrk were subjected to pull down assay with the indicated GST fusion proteins, and bound proteins were analyzed by SDS PAGE followed by IB analysis using an where can i buy pandora charms anti FLAG antibody. (c and d) COS 7 cell lysates transiently cotransfected with plasmids expressing FLAG Hrk and p32(1 282) Myc (c) or transfected with the FLAG Hrk expression plasmid alone (d) were IP with an anti FLAG antibody or with a control IgG, and the immunoprecipitates were subjected to IB analysis with anti c Myc (c) or anti p32 (d), as well as with anti FLAG antibodies. N terminally processed) p32, since this band shows a migration pattern identical with that of p32(74 282) Myc (see Figure 4) Full figure and legend (196K) Binding of p32 and deletion mutants of HrkWe then went on to determine the region of Hrk responsible for binding to p32. 59 constructed a series of deletion mutants lacking one or more of these regions, and expressed these Hrk mutants in COS cells. We could not detect protein expression of some of the mutants, and therefore used for the subsequent binding analysis only those Hrk mutants schematically presented in Figure 3a, whose protein expression was confirmed by immunoblot (IB) analysis (Figure 3b). When these Hrk mutants were tested for binding with GST HA p32, GST Bcl xL, and control GST (fusion) proteins, the results indicated that none of the pre BH3 region, the BH3 domain, the post BH3 region, and the TM domain were required for binding with p32, whereas the BH3 domain was apparently essential for binding with Bcl xL as reported previously.10 Thus, unlike Bcl xL, p32 binds Hrk independent of the BH3 domain, and the post BH3 region of Hrk appears to be responsible for binding with p32. (a and b) Schematic presentation (a) and steady state protein expression (b) of wild type and deletion mutants of Hrk. (c) COS 7 cells were transiently transfected with plasmids expressing the Hrk constructs, and the cell lysates were subjected to pull down assay with the indicated GST fusion proteins. Bound Hrk proteins were detected by IB analysis with an anti FLAG antibody Full figure and legend (121K) The conserved C terminal region of p32 is essential for binding and colocalization with HrkWe next wished to define the region on the part of p32 involved in binding to Hrk. 1 of p32 contains a signal sequence that targets p32 to mitochondria and is removed from the mature p32 protein. 222 highly conserved in different and distant species, namely, from yeast to human.18, 23 To examine the possible contribution of these N and C terminal regions to binding with Hrk, we constructed deletion mutants of p32 that lack the N terminal signal sequence alone (p32(74 or both the signal sequence and the C terminal conserved region (p32(74 (Figure 4a). Two versions (an Myc tag added either to the N terminus or to the C terminus) were created for each of the deletion mutants, expressed in COS cells, and tested for their ability to bind Hrk. As demonstrated in Figure 4b, p32(74 mutants were expressed at higher levels than p32(74 yet only p32(74 mutants but not p32(74 were pulled down by GST FLAG Hrk, irrespective of whether the mutants were tagged at the N terminus or the C terminus. Thus, Hrk binds the mature p32 protein and does not require the N terminal signal sequence for binding. This is also supported by the results of co immunoprecipitation assays presented earlier, since (1) the p32(1 protein expressed in COS cells and co precipitated with Hrk was considered to be processed to the mature form (Figure 2c) and (2) the endogenous, mature p32 protein co precipitated with Hrk (Figure 2d). On the other hand, the results indicated that the highly conserved C terminal region is indispensable for p32 binding to Hrk. The attempt to examine whether the C terminal region of p32 is 'sufficient' for binding to Hrk failed, because the C terminal region alone appeared to be quite unstable and could not be expressed at a detectable level in mammalian cells in our hands. (a) Schematic presentation of the p32 constructs used in this study. (b) COS 7 cells were transiently transfected with plasmids expressing p32(74 or p32(74 tagged with an Myc epitope either at the N terminus (upper panel) or at the C terminus (lower panel). The cell lysates were then used for pull down assay with the indicated GST fusion proteins. Bound p32 mutants were detected by IB analysis with an anti c Myc antibody Full figure and legend (233K) We next investigated the intracellular distribution of the wild type (p32(1 and the deletion mutants of p32 by immunocytochemistry (Figure 5a). Consistent with earlier reports describing that p32 is predominantly a mitochondrial protein,18, 19, 24, 25 the wild type p32 protein showed a typical pattern of mitochondrial staining. In contrast, the p32(74 mutant lacking the mitochondrial signal sequence, tagged either N or C terminally, completely lost the mitochondrial pattern and were instead localized to the nucleus as well as in the cytosol. It was also confirmed that deletion of the C terminal conserved region has no substantial impact on the subcellular localization of p32. In parallel with p32, we also examined the subcellular distribution of Hrk. A previous study indicated that Hrk shows a 'cytoplasmic, granular distribution pattern',10 but the precise subcellular location of Hrk remains unknown. To test the idea that Hrk is localized to mitochondria, we probed Hrk in the presence of a mitochondrial marker, MitoTracker, in the immunocytochemical analysis. As shown in Figure 5b, virtually all signals of Hrk immunostaining colocalized with MitoTracker, indicating that Hrk is predominantly localized to mitochondria. (a) COS 7 cells were transiently transfected with plasmids expressing the indicated p32 constructs, and processed for immunofluorescence analysis using an anti c Myc antibody. (b) Hrk is targeted to mitochondria. COS 7 cells were transiently transfected with a plasmid expressing FLAG tagged Hrk and double stained for FLAG Hrk (green fluorescence) and mitochondria (MitoTracker, red fluorescence). (c) The conserved C terminal region of p32 is essential for the colocalization of Hrk and p32. COS 7 cells were transiently cotransfected with plasmids encoding FLAG Hrk and Myc tagged p32 mutants and processed for immunofluorescence analysis using anti c Myc (green) and anti FLAG (red) antibodies. The arrowheads indicate nuclear margin Full figure and legend (536K) The result that both p32 and Hrk, even when expressed independently from each other, are localized in mitochondria strongly suggested that these proteins interact and form a complex in this organelle within intact cells. We then investigated the effect of Hrk expression on the subcellular distribution of the p32 mutants, which show apparently different subcellular localization from that of Hrk when expressed separately. When expressed alone, p32(74 shows diffuse cytosolic and nuclear pattern as described above (Figure 5a). However, when expressed together with Hrk, a significant proportion of cytosolic p32(74 accumulated in a granular pattern and colocalized with Hrk (Figure 5c). Importantly, p32(74 which we showed to be incapable of binding Hrk, remained diffusely distributed and did not colocalize even when coexpressed with Hrk (Figure 5c). Thus, the results provide unequivocal evidence that p32 and Hrk associate within intact mammalian cells and that this association requires the highly conserved C terminal region of p32. Inhibition of Hrk induced apoptosis by p32 deletion mutants lacking the N terminal mitochondrial signal sequenceThe above results suggest that the p32(74 mutants localized in the cytosol, but not p32(74 mutants incapable of binding Hrk, can compete for Hrk with the endogenous p32 protein localized within mitochondria. So, if interaction with the endogenous p32 is an integral step for Hrk to induce apoptosis, then the p32(74 mutants would inhibit Hrk induced apoptosis in a dominant negative manner. To test this possibility, we examined the effect of p32(74 expression on Hrk induced apoptosis (Figure 6). We first confirmed that transfection mediated expression of FLAG tagged Hrk efficiently induces apoptosis in COS cells, consistent with earlier reports.9, 10 Expression of p32(74 or p32(74 did not have significant effects on the basal level of apoptosis. However, when these mutants were coexpressed together with FLAG Hrk, FLAG Hrk induced apoptosis was significantly reduced by p32(74 but not by p32(74 (Figure 6a). It should be noted that, in the presence of p32(74 the expression level of FLAG Hrk was reproducibly higher than when expressed in its absence (Figure 6b). Since Bcl xL and a pancaspase inhibitor zVAD fmk also showed a similar effect on FLAG Hrk expression (J Sunayama and C Kitanaka, unpublished observation), the Hrk protein may be degraded by an as yet unknown mechanism activated during apoptosis and, conversely, apoptosis inhibition may stabilize and increase Hrk protein expression. We also noticed that the FLAG Hrk expression level tended to be lower when expressed together with p32(74 for which we do not currently have an explanation (Figure 6b).

The inhibitory effect of p32(74 was further confirmed using a different system. Since the expression of FLAG Hrk efficiently suppressed colony formation of U251 human glioma cells, we used this system and asked whether p32(74 could prevent Hrk mediated suppression of colony formation. The results shown in Figure 6c indicate that p32(74 constructs restored colony formation suppressed by FLAG Hrk.

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