Tài liệu Báo cáo khoa học: An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing pdf

An alternative transcript from the death-associated protein kinase locus encoding a small protein selectively mediates membrane blebbing Yao Lin1, Craig Stevens1, Roman Hrstka2, Ben Harrison1, Argyro Fourtouna1, Suresh Pathuri1, Borek Vojtesek2 and Ted Hupp1 Institute of Genetics and Molecular Medicine, Cell Signalling Unit, CRUK p53 Signal Transduction Group, University of Edinburgh, UK Masaryk Memorial Cancer Institute, Brno, Czech Republic Keywords DAPK-1; ERK; membrane blebbing; p53; proteolysis Correspondence T Hupp, Institute of Genetics and Molecular Medicine, Cell Signalling Unit, CRUK p53 Signal Transduction Group, University of Edinburgh, Edinburgh EH4 2XR, UK Fax: +44 131 7773542 Tel: +44 131 7773583 E-mail: ted.hupp@ed.ac.uk (Received 14 January 2008, revised 11 March 2008, accepted 14 March 2008) doi:10.1111/j.1742-4658.2008.06404.x Death-associated protein kinase (DAPK-1) is a multidomain protein kinase with diverse roles in autophagic, apoptotic and survival pathways Bioinformatic screens were used to identify a small internal mRNA from the DAPK-1 locus (named s-DAPK-1) This encodes a 295 amino acid polypeptide encompassing part of the ankyrin-repeat domain, the P-loop motifs, part of the cytoskeletal binding domain of DAPK-1, and a unique C-terminal ‘tail’ extension not present in DAPK-1 Expression of s-DAPK-1 mRNA was detected in a panel of normal human tissues as well as primary colorectal cancers, indicating that its expression occurs in vivo s-DAPK-1 gene transfection into cells produces two protein products: one with a denatured mass of 44 kDa, and a smaller product of 40 kDa Double alanine mutation of the C-terminal tail extension of s-DAPK-1 (Gly296 ⁄ Arg297) prevented production of the 40 kDa fragment, suggesting that the smaller product is generated by in vivo proteolytic processing The s-DAPK-1 gene cannot substitute for full-length DAPK-1 in an mitogenactivated protein kinase kinase ⁄ extracellular signal-regulated kinase-dependent apoptotic transfection assay However, the transfection of s-DAPK-1 was able to mimic full-length DAPK-1 in the induction of membrane blebbing The 44 kDa protease-resistant mutant s-DAPK-1G296A ⁄ R297A had very low activity in membrane blebbing, whereas the 40 kDa s-DAPK1Dtail protein exhibited the highest levels of membrane blebbing Deletion of the tail extension of s-DAPK-1 increased its half-life, shifted the equilibrium of the protein from cytoskeletal to soluble cytosolic pools, and altered green fluorescent protein-tagged s-DAPK-1 protein localization as observed by confocal microscopy These data highlight the existence of an alternative product of the DAPK-1 locus, and suggest that proteolytic removal of the C-terminal tail of s-DAPK-1 is required to stimulate maximally its membrane-blebbing function Death-associated protein kinase (DAPK-1) is a Ca2+ ⁄ calmodulin-regulated serine ⁄ threonine kinase composed of multiple functional domains, including a kinase domain, a calmodulin-binding domain, eight ankyrin repeats, two P-loop motifs, a cytoskeletal binding domain, a death domain, and a C-terminal regulatory tail [1] It has been shown that DAPK-1 is involved in the regulation of distinct processes, Abbreviations GFP, green fluorescent protein; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; TM, tail mutant 2574 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Lin et al including apoptosis, cell survival, and autophagy pathways, with each role depending on the cellular context and the upstream signals [1–5] No apparent defects in developmental cell death were observed in DAPK1-knockout mice [1], thus providing no obvious insights into its stress-regulated functions However, recent research has found that loss or reduced expression of DAPK-1 underlies cases of heritable predisposition to chronic lymphocytic leukemia and the majority of cases of sporadic chronic lymphocytic leukemia [6], suggesting an important role of DAPK-1 in altering the incidence of certain cancer types This is consistent with the ability of DAPK-1 to play a fundamental role in oncogene activation of the p53 tumor suppressor pathway [7] DAPK-1 is relatively large for a protein kinase, and its independent functional domains are involved in various regulatory activities The DAPK-1 kinase domain is required to mediate cytoskeleton remodeling by phosphorylating myosin light chain [8], inhibiting cell migration [9] and inducing membrane blebbing [10] The latter has been characterized as a common morphology correlating with apoptosis or autophagic cell death signals, and the actin–myosin system is considered to be the source of the contractile force underlying the bleb formation [11] Furthermore, microtubule-associated protein 1B interaction with the kinase domain of DAPK-1 stimulates membrane blebbing and autophagy [12] The roles of its other functional domains in its regulatory effects are being characterized For example, the death domain of DAPK-1 forms a docking site for its interaction with extracellular signal-regulated kinase (ERK) [6], and is thus required for DAPK-1’s proapoptotic effect in response to the mitogen-activated protein kinase kinase (MEK) ⁄ ERK signaling pathway [3] Moreover, a germline mutation in the death domain of DAPK-1 has been found to reduce intrinsic oligomerization of the death domain, disrupt the binding of ERK, and thus prevent MEK ⁄ ERK-induced apoptosis [13] The death domain of DAPK-1 also promotes its interaction with the netrin-1 receptor UNC5H2, whose proapoptotic effect when unbound to netrin-1 is partially attenuated in the absence of DAPK-1 [14] The ankyrin-repeat region of DAPK-1 is required for its proper localization to the actin stress fibers [8] and for stable binding with DAPK-1’s ubiquitin E3 ligase, called DAPK-1-interacting protein [4] Recently, it was shown that the leukocyte common antigen-related tyrosine phosphatase interacts with the ankyrin-repeat region of DAPK-1 and dephosphorylates DAPK-1 at pY491 ⁄ 492 to stimulate its proapoptotic and antimigration activities [15] There are many regions ⁄ minido- Functional transcript expressed by DAPK-1 locus mains on DAPK-1 without an ascribed function, and it is likely that further biochemical characterization will result in a greater understanding of the DAPK-1 gene product in autophagic and apoptotic cell signaling Here we report on an mRNA product of the DAPK-1 locus that encodes a small miniprotein (named s-DAPK-1), which shares some domains with full-length DAPK-1: from part of the ankyrin-repeat region, through to part of the cytoskeleton binding domain, and concluding with a unique tail extension of 42 amino acids that is not present in full-length DAPK-1 Unlike DAPK-1, s-DAPK-1 cannot induce apoptosis in response to MEK ⁄ ERK signaling However, s-DAPK-1 can mimic full-length DAPK-1’s ability to promote membrane blebbing The unique C-terminal tail of s-DAPK-1 contains an internal proteolytic processing site whose removal stimulates maximally the membrane-blebbing-promoting effect of s-DAPK-1 These data together identify a novel function for the DAPK-1 locus through the expression of a gene product with a relatively specific role in membrane blebbing Results DAPK-1 is composed of multiple independent minidomains, and in an attempt to determine whether homologous minidomains encoded by alternative genes might exist that compete with or mimic DAPK-1 function, we searched for evidence of the existence of alternatively expressed messages in databases In particular, the ankyrin repeat of DAPK-1 is a potentially versatile protein–protein interaction motif [16], and similar proteins in the human genome might be found that crosstalk to the DAPK-1 pathway Therefore, we evaluated the homology of the ankyrin-repeat region of DAPK-1 with other genes in the human genome using the NCBI nucleotide blast tool One Homo sapiens cDNA was identified: FLJ45958 fis, clone PLACE7011559, from the NEDO human cDNA sequencing project The mRNA of this expression clone starts on intron 13–14 of the DAPK-1 gene and stops on intron 20–21 (Fig 1A) The start codon, ATG, of this expression clone is located on the 10–12th base pairs of exon 15 within the DAPK-1 gene, which makes the translation of this clone in-frame with that of DAPK-1 mRNA After the start codon, this expression clone shares the same sequence as DAPK-1 mRNA through the end of exon 20, as indicated, and its stop codon, TAG, is located on the 124–126th base pairs of intron 20–21 (Fig 1A) Thus, the first 295 amino acids of this 337 amino acid protein are identical to the region of the FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2575 Functional transcript expressed by DAPK-1 locus Y Lin et al Fig The identification of a small transcript from the DAPK-1 locus (A) A schematic map of s-DAPK-1 mRNA in relation to the DAPK-1 gene structure The mRNA of s-DAPK-1 starts in intron 13–14 of the DAPK-1 gene Its coding region starts from the 10th base pair on exon 15 of the DAPK-1 gene, and shares the same splicing as full-length DAPK-1 through the rest of exons 15, 16, 17, 18, 19 and 20 s-DAPK-1’s coding region stops at the 126th base pair of intron 20–21 of the DAPK-1 gene, and the 3¢-UTR extends through the middle of intron 20–21 (B) Comparison of the protein sequences of DAPK-1 and s-DAPK-1 The first 295 amino acids of s-DAPK-1 are identical to amino acids 447–743 of full-length DAPK-1; however, the last 42 amino acids comprise a unique tail (C) Identification of s-DAPK-1 mRNA RT-PCR was performed using the Stratagene QPCR Human Reference Total RNA, and the products were subjected to electrophoresis and staining with ethidium bromide (D) mRNA level test using SYBR Green real-time PCR The relative mRNA level is depicted as a ratio of DAPK-1 ⁄ s-DAPK-1 to actin (E, F) s-DAPK-1, DAPK-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantification in colon carcinoma and rectal carcinoma as compared to normal colonic tissue Colon carcinoma cells, rectal carcinoma cells and their normal healthy tissue counterparts were harvested (1a, carcinoma cells; 4a, normal tissues), and the relative mRNA was quantified using SYBR Green realtime PCR as described previously for the DAPK-1 gene [2] 2576 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Lin et al DAPK-1 protein from residues 447–743, whereas the last 42 amino acids are unique for this product (Fig 1B) These data suggest that this product is highly similar to and may be a splice variant of DAPK-1 Because of its smaller size as compared to full-length DAPK-1, we have named it s-DAPK-1 The transcription of s-DAPK-1 was demonstrated further by RT-PCR using the Stratagene (La Jolla, CA, USA) QPCR Human Reference Total RNA and the primers located on both ends of the coding region of s-DAPK-1 mRNA (Fig 1C) In order to determine the expression of s-DAPK-1, we first compared its mRNA expression with that of DAPK-1 in three widely used tumor cell lines, and we saw a general coincidence between full-length DAPK-1 and s-DAPK-1 mRNA levels (Fig 1D) Next, we set out to determine whether the s-DAPK-1 expression occurs in normal human tissue as well as primary human cancers, rather than just cell lines and cDNA from the NEDO human sequencing project We evaluated the expression of the mRNAs encompassing full-length DAPK-1 and s-DAPK-1 in colorectal carcinomas (1a) and their normal tissue counterpart (4a) using real-time PCR As indicated, DAPK-1 and s-DAPK-1 seem to possess similar mRNA expression profiles throughout the samples (Fig 1E,F) When full-length DAPK-1 was found to be repressed in C18_222_1a tissue, the s-DAPK-1 isoform was also found to be repressed and undetectable (Fig 1E) These data indicate that s-DAPK-1 expression can occur in primary human cancers, and the product of this mRNA was subsequently evaluated as described below Furthermore, s-DAPK1 expression in normal intestinal tissue indicates that its expression is not the result of aberrant splicing, which is known to occur in human cancers To begin functional studies of s-DAPK-1, the s-DAPK-1 cDNA was cloned into a Flag–Myc vector (Fig 2A), which contains an N-terminal Flag tag and a C-terminal Myc tag, and this was followed by expression in HCT116 p53+ ⁄ + cells Two major transfected bands were observed: a 44 kDa upper band, and a 40 kDa lower band (Fig 2B) In order to determine which band corresponded to s-DAPK-1, the C-terminal Myc tag was deleted (Fig 2A) Upon transfection, the same lower protein band was observed in the Flag–s-DAPK-1- and the Flag–s-DAPK-1-Myctransfected cells, whereas the upper band in the Flag– s-DAPK-1 transfection lane was slightly smaller (Fig 2C, lane versus lane 1) This suggests that the depletion of the Myc tag only changes the size of the upper band, and that therefore the upper band represents the ‘full-length’ s-DAPK-1 Functional transcript expressed by DAPK-1 locus Two s-DAPK-1 deletion mutants, Flag-AO (Ankyrin repeat Only) and Flag-TD (Tail Deletion; s-DAPK-1Dtail) were created (Fig 2A) to further investigate why the lower molecular mass protein was observed Upon transfection, the Flag-TD vector produces only one major band (s-DAPK-1Dtail) of lower molecular mass (Fig 2D, lane 3) similar to the 40 kDa lower band produced from the full-length s-DAPK-1 (Fig 2D, lane versus lane 3) This suggests that the lower band might be a cleavage product of the fulllength s-DAPK-1, and that the cleavage signal is within the C-terminal tail extension This is further suggested by the in vitro cleavage assay, in which the purified glutathione S-transferase (GST)–s-DAPK-1 was incubated with HCT116 p53+ ⁄ + cell lysates With increasing amount of cell lysates, GST–s-DAPK-1 was cleaved in vitro at a faster rate than GST alone (Fig 2E), supporting the existence of a protease that cleaves s-DAPK-1 protein in vivo The reason why the cleavage band was not observed in this assay may be the rapid degradation of the purified protein from the cell lysate When subjected to a longer exposure, the blot showed multiple bands below GST–s-DAPK-1, which may mask the actual cleavage band (data not shown) The higher molecular mass protein band ( 54 kDa) might result from a covalent adduct resulting from ubiquitin-like modification; nevertheless, this apparent adduct is dependent upon the integrity of the C-terminal tail extension Since it had been confirmed that the cleavage is within the tail, we next investigated sites within the tail that are the critical targets for the cleavage Because the transfected Flag-TD vector (s-DAPK-1Dtail) is similar to the in vivo cleaved form of Flag–s-DAPK-1 in size, five tail mutants (TMs) of s-DAPK-1 were created to screen the first 10 amino acids on the tail for proteolytic susceptibility (Fig 3A) Upon transfection, only s-DAPK-1G296A ⁄ R297A (TM1) exhibited a reduced proteolytic band, and s-DAPK-1N298A ⁄ L299A (TM2) showed a weakened cleavage band (Fig 3B) These data suggest that the first two amino acids of the tail are critical for proteolytic susceptibility, and that the third and fourth amino acids are involved in the regulation of this cleavage This also further fine-maps the site of cleavage, and indicates that the tail deletion (s-DAPK-1Dtail) may be used as a mimic of the in vivo processed form of full-length s-DAPK-1 s-DAPK1H300A (TM3) surprisingly produced a specific shift in size under denaturing conditions, suggesting that the modification of the fifth amino acid on the tail may alter its secondary structure in denaturing polyacrylamide gels or might yield an undefined covalent adduct (Fig 3B, lane 4) FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2577 Functional transcript expressed by DAPK-1 locus Y Lin et al Fig Identification of a proteolytic cleavage within the C-terminal tail of s-DAPK-1 protein (A) A schematic diagram of the Flag–Myc vector with the s-DAPK-1 clone and its mutants created by site-directed mutagenesis The vector encoding the s-DAPK-1Dtail with a 42 amino acid tail deletion is named Flag-TD (B–D) Transfected s-DAPK-1 and its mutants identified a cleavage within its tail HCT116 p53+ ⁄ + cells were transfected with the respective vectors, as indicated, for 24 h prior to harvesting Expression of the ectopically expressed s-DAPK-1 and its mutants was detected using an antibody to Flag (Sigma) (E) In vitro cleavage of purified GST–s-DAPK-1 Recombinant GST–s-DAPK-1 was purified from Bl21 cells and incubated at 30 °C with increasing amounts of HCT116 p53+ ⁄ + cell lysates (0, 1, 5, 10 and 20 lL) as indicated The sample mixtures after in vitro cleavage were subjected to immunoblotting 2578 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Lin et al Functional transcript expressed by DAPK-1 locus Fig Identification of the critical sites for proteolytic cleavage of the C-terminal tail of s-DAPK-1 (A) A schematic diagram of the tail mutants of s-DAPK-1 created by sitedirected mutagenesis (B) Expression of the tail mutants of s-DAPK-1 HCT116 p53+ ⁄ + cells were transfected with the respective vectors, as indicated, for 24 h prior to harvesting Expression of the s-DAPK-1 tail mutants was detected by immunoblotting (C) Cleavage of the tail of s-DAPK-1 is not inhibited by common protease inhibitors HCT116 p53+ ⁄ + cells were transfected with the Flag–s-DAPK-1 vector for 24 h and treated with the indicated protease inhibitors h prior to harvesting The Flag–s-DAPK-1 protein was detected by immunoblotting The proapoptotic effect in response to MEK ⁄ ERK signaling and the membrane-blebbing-promoting effect upon transfection are two well-characterized functions of DAPK-1 [3,10,13] Therefore, we set out to define the role of s-DAPK-1 in these two pathways We used, as expressed constructs, full-length s-DAPK-1, s-DAPK1Dtail, and s-DAPK-1G296AR297A, which allowed us to evaluate whether the tail contributes to the function of the s-DAPK-1 protein Unlike DAPK-1, s-DAPK-1 does not induce poly (ADP-ribose) polymerase (PARP) cleavage in response to the MEK ⁄ ERK signal input (Fig 4A, lane versus lane 5) However, transfection of Flag–s-DAPK-1 was able to cause significant membrane blebbing, to levels similar to those caused by fulllength DAPK-1, although the effect was weaker (Fig 4B) Given the biological activity of s-DAPK-1 in the membrane-blebbing assay, we evaluated the activity of the mutant with the tail deletion (Flag-TD; s-DAPK1Dtail) and the protease-resistant substitution (FlagTM1; s-DAPK-1G296AR297A) As compared to full-length s-DAPK-1, the s-DAPK-1G296AR297A showed a reduced membrane-blebbing effect (Fig 4C), whereas s-DAPK-1Dtail was almost as active as fulllength DAPK-1 (Fig 4C) These data indicate that the ‘tail’ of s-DAPK-1 has a negative regulatory function with regard to s-DAPK-1 activity, and that its removal serves to enhance the membrane-blebbing effect of s-DAPK-1 To determine the mechanism that underlies the effect of the tail on the membrane-blebbing-promoting ability of s-DAPK-1, the localization and half-lives of the full-length s-DAPK-1, s-DAPK-1Dtail and s-DAPK1G296AR297A were examined As compared to DAPK-1, s-DAPK-1 shows more specific localization in the cytoplasm (Fig 5A) s-DAPK-1Dtail predominantly localizes around the nucleus, and s-DAPK1G296AR297A spreads throughout the cytosol and tends to form some ‘aggregating bodies’ (Fig 5) Moreover, the half-life of s-DAPK-1Dtail is much longer than those of s-DAPK-1 and s-DAPK-1G296AR297A (Fig 6A–D), suggesting that the increased membraneblebbing function of s-DAPK-1Dtail is due to its slower FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2579 Functional transcript expressed by DAPK-1 locus Y Lin et al Fig The C-terminal tail of s-DAPK-1 negatively regulates its membrane-blebbing function (A) s-DAPK-1 does not induce apoptosis in response to MEK ⁄ ERK signaling HEK293 cells were transfected with the respective vectors, as indicated, for 24 h prior to harvesting PARP and PARP cleavage were detected with a PARP-specific antibody (Cell Signalling) (B) s-DAPK-1 induces membrane blebbing A375 cells were transfected with the respective vectors as indicated, and evaluated for membrane blebbing in transfected cells as described previously [10] The top panel (B) shows the normal (1) and the blebbing (2) morphology (C) The C-terminal tail modulates membrane blebbing by s-DAPK-1 A375 cells were transfected with the respective vectors as indicated (wt, TM1, and TD; s-DAPK-1Dtail) and evaluated for membrane blebbing in transfected cells as described previously [10] The top panel (B) shows the normal (1) and blebbing (2) morphology The bar graph in the bottom panels of (B) and (C) summarizes the mean percentage of blebbing cells upon each transfection Each experiment was repeated four times degradation (Fig 6B) Furthermore, upon chemical subcellular fractionation based on differential protein solubility, s-DAPK was found to localize in both the ‘insoluble’ cytoskeletal and soluble cytosolic fractions (Fig 5B), whereas the s-DAPK-1Dtail equilibrium was shifted more into the soluble cytosolic fraction (Fig 5C) Discussion DAPK-1 is a stress-regulated kinase whose downstream functions are linked to a variety of diverse 2580 signaling pathways, including ERK kinase activation, autophagic signaling, and oncogene-mediated p53 transcriptional responses DAPK-1 is also regulated by tumor necrosis factor signaling, p90 ribosomal S6 kinase (RSK), and leukocyte common antigen-related phosphatase, which alter the specific activity of the kinase as a prosurvival or proapoptotic factor Although the DAPK-1 protein is now known to be regulated post-translationally, the gene is also subject to methylation, which has the potential to reduce the specific activity of DAPK-1 [6] In this work, we have identified another function of the DAPK-1 locus: it can express a message whose product possesses part of DAPK-1’s ankyrin-repeat region, P-loop, and cytoskeletal binding domain, and a unique tail of 42 amino acids encoded by intron 20–21 of the DAPK-1 gene In our examination of DAPK-1 and s-DAPK-1 expression using real-time PCR, we found a significant correlation in their expression, whether using cancer cell lines or normal human tissues, suggesting that mRNA from the locus is coordinately produced Future work will be required to understand the regulation of the translation of these mRNAs and whether stress-regulated signaling pathways regulate these two proteins differently in cell growth control Despite the many functions attributed to DAPK-1, the two standard cellular assays for defining its function include proapoptotic pathways and membrane blebbing Therefore, we have examined the ability of the s-DAPK-1 protein to play a role in these two processes We found that although s-DAPK-1 cannot induce apoptosis in response to the activated MEK ⁄ ERK signal like DAPK-1, it can mimic DAPK1 and induce membrane blebbing A function was also attributed to the unique tail of s-DAPK-1: it can regulate the localization and half-life of the protein and FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Lin et al Functional transcript expressed by DAPK-1 locus Fig The C-terminal tail of s-DAPK-1 regulates its localization (A) Confocal microscopy A375 cells were transfected with the respective vectors as indicated [GFP control, GFP-wt s-DAPK, GFP-TM1, GFP-TD (s-DAPK-1Dtail), and HA-DAPK-1] The localizations of GFP and GFPtagged proteins were detected under the microscope HA–DAPK-1 protein expression was detected using an antibody to HA tag (B, C) Subcellular protein fractionation Chemical fraction of cell pellets after FLAG–s-DAPK transfection into a cytosolic (A) and cytoskeletal (B) fractions for Flag–s-DAPK or Flag–s-DAPK-1Dtail (TD) as indicated in Experimental procedures FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS 2581 Functional transcript expressed by DAPK-1 locus Y Lin et al Fig The C-terminal tail of s-DAPK-1 regulates its half-life (A–D) The half-life of s-DAPK-1 is regulated by its C-terminal tail HCT116 p53+ ⁄ + cells were transfected with the respective vectors as indicated for 24 h, in combination with cycloheximide treatment at the indicated times, prior to harvesting Expression of the Flag-tagged proteins was evaluated by immunoblotting can be subjected to proteolytic cleavage in cells Presumably, the tail has evolved to relocalize s-DAPK1 to mediate its rapid degradation, which would greatly attenuate its membrane-blebbing function Signals that produce proteolytic cleavage would in turn reduce its degradation (Fig 6) and allow it to function fully as a membrane-blebbing factor DAPK-1 has been shown to induce membrane blebbing and promote the formation of actin stress fibers and disassembly of focal adhesions [17] These biological events can occur in cooperation with microtubuleassociated protein 1B [12], for which the ability of DAPK-1 to phosphorylate myosin light chain [8] and tropomyosin-1 [18] are considered to be important However, it was also shown that the ankyrin-repeat region deletion mutant of DAPK-1 mislocalized to focal contacts and lost its ability to induce morphological changes [8], indicating a functional role of this region in DAPK-1’s activity This might explain the membrane-blebbing-promoting effect of s-DAPK-1, as it shares a portion of the ankyrin-repeat region of DAPK-1 However, the functional significance of the s-DAPK-1-induced membrane blebs is not clear, as s-DAPK-1 cannot induce MEK ⁄ ERK-stimulated apoptotic signals (Fig 5A) A recent study has provided a novel insight into membrane blebbing [19]; it was shown that membrane blebbing is due to the reassembly of the contractile cortex Therefore, distinct from the alternative models showing that membrane blebbing is linked to autophagic or cell death pathways, membrane blebbing may also be part of a normal cell division processes such as cytokinesis Considering that ankyrin B plays an important role in the membrane-blebbing process [19], DAPK-1 and s-DAPK-1 may be able to interact with ankyrin B via their ankyrin repeats and thus promote membrane blebbing Although these data provide an explanation for the significance of the ankyrinrepeat region of DAPK-1 in inducing morphological changes, they not necessarily indicate that DAPK-1or s-DAPK-1-induced membrane blebbing is part of a normal cell division cycle Considering that physiologi2582 cal membrane blebbing is a transient process [19], it also remains possible that DAPK-1 and s-DAPK-1 arrest the cells at the blebbing stage and thus halt the cell division cycle Therefore, the actual biological significance of the s-DAPK-1- and DAPK-1-induced membrane blebbing requires further investigation Experimental procedures Cell culture and harvest, plasmids and transfection, and treatment HEK293 (human embryonic kidney cell line) and A375 (human melanoma) cells were cultured in DMEM medium, and HCT116 (human colon carcinoma) cells were cultured in McCoy medium The medium was supplemented with 10% fetal bovine serum and a penicillin and streptomycin mixture at 37 °C with 5% CO2 in a humidified atmosphere In a typical experiment, 106 cells were seeded into a 10 cm tissue culture plate and left for at least 24 h to attach to the bottom of the container Before harvesting, cells were first washed twice with NaCl ⁄ Pi and then scraped into mL of NaCl ⁄ Pi The HA–DAPK-1 was a kind gift from A Kimchi (Weizmann Institute, Rehovot, Israel) s-DAPK1 was cloned into the Flag–Myc vector from Sigma (Poole, UK), and the mutants were created using the Quickchange site-directed mutagenesis kit from Stratagene GST–sDAPK-1 was cloned into the pDEST-15 gateway GST vector from Invitrogen The primers for cloning and mutations are available upon request Prior to transfection, Lipofectamine ( lLỈlg)1 DNA) was added to Optimum medium without fetal bovine serum After a incubation, the mixture was added to the DNA constructs, and after a 30 incubation at room temperature, the whole solution was added to the cells The translation inhibitor cycloheximide from Supleco (Bellefonte, PA, USA) was used at a concentration of 10 lgỈmL)1 Protein analysis Proteins were extracted by lysing the cells with lysis buffer (1% NP40, 0.15 m NaCl, 50 mm Tris, pH 7.5, mm FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Lin et al dithiothreitol and 1· protease inhibitor mixture), and the protein concentrations were determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA) Immunoprecipitation, protein separation by SDS ⁄ PAGE and detection by immunoblotting were done as previously described [2] The following antibodies were used: anti-HA (Covance, Princeton, NJ, USA), anti-GST and anti-Flag (Sigma), and anti-PARP (Cell Signal) The ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, La Jolla, CA, USA) was used to extract proteins from mammalian cells according to their cytosolic or cytoskeletal subcellular localization The kit was used in accordance with the manufacturer’s recommendations All extraction buffers contained protease inhibitors, and all steps were carried out at °C unless stated The cytosolic and cytoskeletal fractions were stored at )70 °C and analyzed by immunoblotting RNA extraction, reverse transcription, PCR and real-time PCR mRNA was extracted from cells and tissue (obtained with local ethical permission from the Masaryk Institute ethics committee) using the Qiagen RNeasy Mini kit, following the manufacturer’s suggested procedures The optional step of DNase treatment using the Qiagen RNase-free DNase set was also included After the extraction, RT-PCR was performed using the Omniscript RT kit from Qiagen (Valencia, CA, USA) and pfu polymerase from Stratagene, following the manufacturer’s suggested protocols Real-time PCR was performed using the Qiagen QuantiTect SYBR Green one-step PCR kit, following the manufacturer’s suggested protocols The actin primers were as follows: forward, 5¢-CTACGTCGCCCTGGACTTCGAGC-3¢; reverse, 5¢-GATGGAGCCGCCGATCCACACGG-3¢ The DAPK1 primers were as follows: forward, 5¢-CGAGGTGA TGGTGTATGGTG-3¢, reverse, 5¢-CTGTGCTTTGCTGG TGGA-3¢ The s-DAPK-1 primers were as follows: forward, 5¢-CGTCTCTCCAGCAGGTGTT-3¢; reverse, 5¢-TA AGGCCACAGGGTCCAGTA-3¢ Immunostaining and membrane-blebbing assay A375 cells were analyzed by immunostaining and membrane blebbing Twenty-four hours post-transfection, cells were fixed with 4% paraformaldehyde in NaCl ⁄ Pi for 10 min, washed, and blocked with antibody dilution buffer (3% BSA in NaCl ⁄ Pi) for h For the non-green fluorescent protein (GFP)-tagged proteins, the transfected cells were then visualized using HA.11 antibody (Covance) and antibody to Flag (Sigma) After incubation with the appropriate primary antibodies for h, cells were washed with NaCl ⁄ Pi, stained with mouse Alexa488-conjugated secondary antibody, and mounted for observation by immunostaining or by examining Functional transcript expressed by DAPK-1 locus membrane-blebbing morphology using a Leica fluorescent microscope For immunostaining, the transfected cells were incubated with Topro-3 from Invitrogen (1 : 1000 in NaCl ⁄ Pi) for 15 at 37 °C before mounting In membrane-blebbing assays, 300 transfected cells were counted upon each transfection, and each experiment was repeated four times In vitro cleavage of bacterial purified protein The GST–s-DAPK-1-transformed BL21 cells were induced with arabinose for h and lysed with 0.2% Triton in NaCl ⁄ Pi The GST–s-DAPK-1 protein was then extracted from the lysate using glutathione–Sepharose (GE Healthcare, Amersham, UK) and eluted with 50 mm glutathione For in vitro cleavage assays, lL of the purified GST fusion protein was incubated at 30 °C with various amounts of lysate, as mentioned above, for 30 The reaction was then stopped by adding SDS sample buffer, and the mixture was subjected to immunoblotting Acknowledgements B Vojtesek and R Hrstka are funded by grants 301 ⁄ 05 ⁄ 0416 and 301 ⁄ 08 ⁄ 1468 from GACR and grant LC06035 T Hupp is funded by grants from Cancer Research UK References Bialik S & Kimchi A (2006) The death-associated protein kinases: structure, function, and beyond Annu Rev Biochem 75, 189–210 Lin Y, Stevens C & Hupp T (2007) Identification of a dominant negative functional domain on DAPK-1 that degrades DAPK-1 protein and stimulates TNFR-1-mediated apoptosis J Biol Chem 282, 16792–16802 Chen CH, Wang WJ, Kuo JC, Tsai HC, Lin JR, Chang ZF & Chen RH (2005) Bidirectional signals transduced by DAPK–ERK interaction promote the 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120, 3666–3677 Charras GT, Hu CK, Coughlin M & Mitchison TJ (2006) Reassembly of contractile actin cortex in cell blebs J Cell Biol 175, 477–490 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS ... plays an important role in the membrane- blebbing process [19 ], DAPK -1 and s-DAPK -1 may be able to interact with ankyrin B via their ankyrin repeats and thus promote membrane blebbing Although these... membrane- blebbing assay, we evaluated the activity of the mutant with the tail deletion (Flag-TD; s-DAPK1Dtail) and the protease-resistant substitution (FlagTM1; s-DAPK-1G296AR29 7A) As compared... to the activated MEK ⁄ ERK signal like DAPK -1, it can mimic DAPK1 and induce membrane blebbing A function was also attributed to the unique tail of s-DAPK -1: it can regulate the localization and