Introduction

Caspases are key players in apoptosis, yet the mechanisms that regulate their activity and function are still largely obscure. We are investigating the crosstalk between non-receptor tyrosine kinases, central transducers of the proliferative response, and caspases and whether these two families of proteins can modulate each other's function through their enzymatic activities. We have previously reported that caspases can directly cleave c-Abl non-receptor tyrosine kinase modulating its cellular localization and its function (Barilà et al, 2003). Conversely, the overexpression of some kinases protects cells from apoptosis (Hunter, 2000). Recent studies have shown that, during cellular proliferation, Akt and MAPK switch-off the activities of Caspase-9 (Cardone et al, 1998; Allan et al, 2003), Caspase-8 and Caspase-3 (Alvarado-Kristensson et al, 2004) by direct Ser/Thr phosphorylation. However, the role of tyrosine phosphorylation in the control of caspase activity remains almost totally underinvestigated.

The Src family of non-receptor tyrosine kinases has been reported to modulate Fas-signaling. Interestingly, defective expression of the SHP-1 tyrosine phosphatase in lymphoid cells blocks Fas-mediated apoptosis (Su et al, 1995), while in neutrophils SHP-1 can bind death receptors and block cytokine-induced antiapoptotic signaling (Daigle et al, 2002) enforcing the idea that tyrosine dephosphorylation of specific substrates is a prerequisite for death receptor-induced apoptosis.

Fas (CD95/APO-1) is a transmembrane protein belonging to the tumor necrosis factor superfamily. Upon binding of Fas ligand or agonistic antibodies, the Fas receptor recruits several cytosolic proteins to form the death-inducing signaling complex (DISC). This is necessary to catalyze dimerization, activation (Boatright et al, 2003; Chang et al, 2003; Donepudi et al, 2003) and processing (Muzio et al, 1996; Medema et al, 1997) of Procaspase-8 to form the Caspase-8 tetramer, composed of two p18 and two p10 subunits (Blanchard et al, 1999; Watt et al, 1999), and initiate the caspase cascade. Procaspase-8 activation is absolutely required to trigger this apoptotic response (Juo et al, 1998) and its catalytic activity has to be tightly regulated to avoid inappropriate activation and undesired cell death (Peter, 2004).

To investigate the role of Src family kinases in the control of caspases, we tested whether non-receptor tyrosine kinases impinge on Fas-signaling directly targeting Caspase-8. We identified Caspase-8 as a novel substrate of Src tyrosine kinase. Src directly phosphorylates Procaspase-8 on Tyr380 and prevents its activation, and therefore its proapoptotic function upon Fas-receptor stimulation. The Src family of non-receptor tyrosine kinases plays an important role in the transduction of the proliferative signal triggered by growth factors like PDGF, EGF and HGF (Bromann et al, 2004). More importantly, Src kinase activity is upregulated in several epithelial and nonepithelial cancers (Frame, 2002). Here, we show that endogenous Caspase-8 is tyrosine phosphorylated upon EGF receptor stimulation. Moreover, EGF impairs Fas-induced apoptosis, most likely through Caspase-8 phosphorylation, pointing to Caspase-8 tyrosine phosphorylation as a novel mechanism to suppress apoptosis. Remarkably, Caspase-8 is phosphorylated on Tyr380 also when Src kinase activity is pathologically upregulated, such as in colon cancer, highlighting Tyr380 phosphorylation as a potential molecular strategy of apoptosis suppression adopted by tumor cells.

Results

Src kinase triggers Caspase-8 tyrosine phosphorylation

To test whether Caspase-8 could be a substrate for non-receptor tyrosine kinases, we transiently transfected Caspase-8 in human embryo kidney (HEK) 293 cells, together with several constructs encoding different forms of Src and of Abl kinases. Since overexpression of Caspase-8 in mammalian cells is sufficient to trigger its own activation and cell death, all transfections were performed in the presence of the strong caspase inhibitor CrmA (Tewari and Dixit, 1995; Zhou et al, 1997). The constitutively active Src, SrcY527F, but not its kinase inactive counterpart, caused a strong tyrosine phosphorylation of cotransfected Caspase-8, as shown by immunoblotting (Figure 1, right panel) and in immunoprecipitation experiments (Figure 1, left panel). Interestingly, neither the constitutively active mutant of c-Abl, Abl-PP, nor the leukemiogenic form of Abl, Bcr-Abl, could phosphorylate Caspase-8, suggesting that Caspase-8 is specifically phosphorylated by Src.

Tyrosine phosphorylation protects cells from Fas-induced apoptosis

Caspase-8 is absolutely required for Fas-induced cell death (Juo et al, 1998) and defective Caspase-8 activity results in the impairment of this apoptotic response. To test whether tyrosine phosphorylation could affect Caspase-8 activity and function, HeLa cells, which are sensitive to Fas, were transfected with a construct encoding the constitutively active form of Src, SrcY527F, along with a plasmid encoding GFP to allow detection of transfected cells. SrcY527F overexpression drives tyrosine phosphorylation of endogenous Caspase-8 (Figure 2A). Interestingly, SrcY527F but not its kinase inactive counterpart, SrcY527F-kin^-, decreases Fas-induced apoptosis, similarly to the strong caspase viral inhibitor CrmA, indicating that Src activity is necessary to mediate its antiapoptotic effect (Figure 2B).

To further explore whether Src-induced resistance to Fas could be directly linked to Caspase-8 phosphorylation, we analyzed the activation of Caspase-8, upon Fas-stimulation, in HeLa cells transfected with the constitutively active Src. SrcY527F overexpression delayed Fas-induced Caspase-8 activation, measured as its ability to cleave its substrate peptide IETD, to an extent remarkably similar to the strong caspase viral inhibitor CrmA (Figure 2C).

These data suggest that tyrosine phosphorylation affects Caspase-8 proapoptotic function.

Src phosphorylates Caspase-8 on Tyr380

To identify Src-induced tyrosine phosphorylated site(s) on Caspase-8, a Myc-tagged version of Caspase-8 was expressed in HEK293 cells, cotransfected with SrcY527F, and immunoprecipitated. The band corresponding to immunoprecipitated Caspase-8 was visualized by Coomassie staining, recovered from SDS–PAGE gel, and digested with trypsin. The peptide mixture was then treated or not treated with alkaline phosphatase and the phosphorylation profile analyzed by MALDI-TOF mass spectrometry. One phosphopeptide was isolated, corresponding to amino acids 368–391, containing a single phosphate group. The identity of the phosphopeptide was further confirmed by the presence of a methionine residue which, when oxidated, caused a further mass shift of 16 Da. The only tyrosine residue within this peptide is the Tyr380 of human Caspase-8 (Figure 3A). To further investigate whether Tyr380 was the major Src phosphorylation site in Caspase-8, we tested a mutant where Tyr380 was mutated to Phe (Caspase-8-Y380F) in cotransfection assays with SrcY527F or with SrcSH3, another constitutively active form of Src (data not shown). Mutation of Tyr380 virtually abrogated tyrosine phosphorylation of Caspase-8, supporting the notion that Tyr380 is the major Src phosphorylation site (Figure 3B).

Interestingly, Tyr380 lies in the linker sequence that separates the large subunit and the small subunit, between the two cleavage residues D374 and D384 (Figure 3C). This region is removed upon activation and it is therefore not part of the structures of the p18–p10 tetrameric complex (Blanchard et al, 1999; Watt et al, 1999). No structural information is currently available on this linker sequence and its function in Caspase-8 regulation remains speculative.

We raised a monoclonal antibody against a phosphopeptide containing phospho-Tyr380. Indeed, this antibody detected Caspase-8 only after Src phosphorylation, whereas mutation of Tyr380 to Phe abolished recognition, demonstrating that the antibody specifically reacts with the phosphorylated site (Supplementary Figure S1).

Src directly phosphorylates Caspase-8 on Tyr 380 and this event impairs Caspase-8 activity in yeast

The yeast Schizosaccharomyces pombe does not express endogenous tyrosine kinases and caspases (Superti-Furga et al, 1993; Silke et al, 2001), and can therefore be used as an in vitro system to address the question whether exogenous Src directly phosphorylates exogenous Caspase-8. Owing to the lack of the physiological regulator Csk, Src is constitutively active in yeast and its activity triggers cell death (Superti-Furga et al, 1993). We observed that Caspase-8 is also highly toxic, depending on its enzymatic activity (data not shown). To allow the expression of high levels of proteins, we coexpressed Src and Caspase-8 under the control of inducible promoters, as previously reported (Superti-Furga et al, 1993). Caspase-8 accumulation in yeast drives its autoprocessing, leading to the ordered production of the p10 (small) and p43 (DEDs-p18) subunits first and then to the release of the p18, detectable by immunoblots with specific antibodies, similarly to what described in the mammalian cells (Figure 4A). Caspase-8 was immunoprecipitated and processed for mass-spectrometry, as described in Figure 3A. Again, Tyr380 was identified as the major phosphorylated residue (data not shown), indicating that Src kinase can directly phosphorylate Caspase-8. The absence of endogenous tyrosine kinases and caspases in S. pombe provides a perfect naïve environment in which to test the effect and the crosstalk of these heterologous proteins (Superti-Furga et al, 1993) on Caspase-8 activity and function. Extracts from S. pombe at different times of protein expression induction, transfected with Src or with the empty vector control along with Caspase-8, were processed for immunoblot analysis using an anti-Caspase-8 antibody raised against the p18 subunit. This antibody detects both the Procaspase-8 and its cleaved p18 subunit. Caspase-8 expression is controlled by an inducible promoter, which allows the detection of Caspase-8 protein by immunoblotting around 10–11 h of induction. At this time we can detect only the presence of Procaspase-8, which further accumulates and is processed and activated at 12, 13 and 14 h. Interestingly, the amount of Procaspase-8 as well as of its cleavage products dramatically decrease at 16 h of induction due to its high toxicity and to consequent cell death (Supplementary Figure S2A). The expression of Src triggers Caspase-8 phosphorylation (data not shown) and results in the delayed accumulation of the p18 subunit at 13 h of induction, supporting the idea that Src kinase activity interferes with Caspase-8 processing and activation (Figure 4B). To further confirm this hypothesis, we analyzed Caspase-8 activation in the same extracts, measured as its ability to cleave its substrate peptide IETD (Figure 4C and Supplementary Figure S2B). Caspase-8 activity starts to be detectable at 12 h of expression, according to the appearance of its processing products, peaks between 13 and 14 h, and dramatically decreases at 16 h, consistently with the decrease of its expression levels (Supplementary Figure S2). Src expression significantly delays Caspase-8 activation at 12 and at 13 h of induction (Figure 4C and Supplementary Figure S2B). These experiments allowed us to conclude that tyrosine phosphorylation impairs Caspase-8 activity in this system.

Caspase-8-wt and Caspase-8-Y380F are equally toxic (Figure 4D and E) indicating that Tyr380Phe mutation does not affect per se Caspase-8 activity. Interestingly, despite its own toxicity, Src coexpression slightly relieved the yeast from Caspase-8 toxicity, suggesting that phosphorylation modulates Caspase-8 activity. Src protective effect requires the phosphorylation of Tyr380 of Caspase-8, since Src coexpression failed to ameliorate the Caspase-8-Y380F toxicity (Figure 4D). Overall, this system allowed us to clarify that Src directly phosphorylates and inactivates Caspase-8 in vivo (data not shown and Figure 4).

Tyrosine phosphorylation modulates Caspase-8 processing and activity in mammalian cells

Fas-receptor stimulation leads to Caspase-8 recruitment to the DISC, dimerization and processing (Boatright et al, 2003; Chang et al, 2003; Donepudi et al, 2003).

Our observation that Caspase-8 activity is downregulated by Src expression in yeast, where Fas-receptor is absent, suggests that Src effect on Caspase-8 activity does not involve its recruitment to the DISC. It has been previously shown that incubation of cell extracts in the presence of Sodium Citrate triggers Procaspase-8 dimerization and activation (Boatright et al, 2003). To investigate whether tyrosine phosphorylation could modulate Procaspase-8 dimerization, protein extracts from HeLa cells transfected with SrcY527F or with an empty vector as a control, were incubated in the presence or in the absence of Sodium Citrate to induce Procaspase-8 dimerization in the presence of biotinyl-VAD-fmk, which labels caspases' active sites. Labeled active Procaspase-8 dimers were then immunoprecipitated with agarose-conjugated streptavidin beads and analysed by Western blot with anti-Caspase-8 antibody. This assay allows the detection of active Procaspase-8, which correlates with its ability to dimerize. Src activity does not affect the amount of biotinyl-VAD-fmk-labelled-Procaspase-8, activated by Sodium Citrate, suggesting that tyrosine phosphorylation does not modulate dimerization. Using our monoclonal phospho-specific antibody that selectively recognizes Tyr380 phosphorylated Caspase-8 (Supplementary Figure S1), we could show that indeed phosphorylated Caspase-8 is able to dimerize (Figure 5A).

We therefore investigated whether tyrosine phosphorylation could affect Caspase-8 processing upon Fas-receptor stimulation. In this pathway, processing is essential to get a stable active Caspase-8 tetramer and to allow its release from the DISC and subsequent cleavage of cytoplasmic substrates, such as executioner caspases. Extracts from Fas-stimulated HeLa cells transfected with SrcY527F or with the empty vector control, were processed for immunoblot analysis using an anti-Caspase-8 antibody raised against the p18 subunit. The expression of SrcY527F results in the delayed accumulation of the p18 subunit (Figure 5B). The appearance of the p18 subunit can be evaluated as a marker for Caspase-8 activation. Indeed, Src expression reduces the ability of the p18, generated upon Fas-receptor stimulation, to bind biotinyl-VAD-fmk (Figure 5C). Overall, these experiments support the idea that Src kinase activity interferes with Caspase-8 processing and activation.

Phosphorylation on Tyr380 impairs Caspase-8 activation upon Fas receptor stimulation

To investigate the effect of Tyr380 phosphorylation on Caspase-8 activity, we chose the Jurkat Caspase-8^-/- cell line. These cells do not express Caspase-8 and therefore do not undergo apoptosis upon Fas-receptor stimulation, yet the reconstitution of Caspase-8 expression restores the sensitivity to Fas-induced apoptosis (Juo et al, 1998). Since Caspase-8 overexpression triggers its own activation, which induces cell death per se, these experiments require the expression of very low levels of transfected Caspase-8, which allow the detection of Fas-induced apoptosis. Caspase-8-wt and Caspase-8-Y380F restore Fas sensitivity to the same extent, confirming that the substitution of Tyr380 with Phe does not affect per se Caspase-8 function. Importantly, coexpression of the constitutively active Src, SrcY527F, suppressed Fas-induced apoptosis in cells reconstituted with Caspase-8 wt, but not in those reconstituted with Caspase-8-Y380F, indicating that Src-mediated protection requires Caspase-8 Tyr380 phosphorylation (Figure 5D) and suggesting that the phosphorylation of Tyr380 modulates Caspase-8 activity and function.

EGF triggers endogenous Caspase-8 phosphorylation and counteracts Fas-induced apoptosis

Endogenous Src kinase activity is tightly regulated and is induced upon different stimuli. To further investigate whether the activation of endogenous Src could trigger Caspase-8 phosphorylation, HeLa cells were treated with EGF, which directly activates Src kinase (Figure 6A). Immunoblotting with anti-phosphotyrosine antibodies on immunoprecipitated Caspase-8 showed that EGF treatment triggered Caspase-8 phosphorylation (Figure 6A). We further asked whether Caspase-8 phosphorylation could affect Fas sensitivity in this context. Exposure to EGF caused a significant delay in Fas-induced apoptosis of HeLa cells (Figure 6B). Interestingly, EGF failed to protect cells stably transfected with a kinase defective version of Src (SrcY527F-kin^-) from Fas-induced apoptosis, suggesting that the antiapoptotic effect of EGF relies on Src kinase activity (Figure 6B) and, most likely, on Caspase-8 phosphorylation.

Caspase-8 is phosphorylated on Tyr380 in colon cancer

We reported that the activation of Src in a physiological situation such as EGF treatment triggers Caspase-8 phosphorylation. We therefore asked whether Caspase-8 may be tyrosine phosphorylated in those pathological situations where Src kinase is aberrantly upregulated such as different types of tumors (Frame, 2002). We speculated that tyrosine phosphorylation of Caspase-8 may represent a new mechanism through which transformed cells evade apoptosis and promote cancer. We analyzed the profile of Caspase-8 in colonic adenocarcinomas from 23 patients. While the levels of Src protein in cancer mucosa and in the adjacent normal mucosa are comparable (Figure 7A), the kinase activity of Src is significantly increased in about 80% of the tumors (Figure 7B and C, Supplementary Figure S3 and Supplementary Table), according to previous reports from other laboratories (Allgayer et al, 2002). More interestingly, we were able to immunoprecipitate Caspase-8 with anti-phosphotyrosine antibodies, selectively from cancer mucosa (Figure 7B). In the reverse experiment, we could detect tyrosine-phosphorylated Caspase-8 upon immunoprecipitation with anti-Caspase-8 antibodies and immunoblotting with anti-phosphotyrosine antibodies (Figure 7C). We revealed the presence of tyrosine-phosphorylated Caspase-8 in 19 out of 23 analyzed patients (Supplementary Table). Using our phospho-specific antibody, which selectively recognizes phosphorylated Tyr380 (Supplementary Figure S1), we were able to show that Caspase-8 is phosphorylated on Tyr380 in primary colon cancer (Figure 7D, Supplementary Figure S3 and Supplementary Table).

To further investigate whether Caspase-8 phosphorylation on Tyr380 could modulate Caspase-8 activity in human colon cancer, we took advantage of a human colon cancer cell line previously selected for being resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, named DLD1/TRAIL-R (Zhang et al, 2005). These cells have been shown to express low levels of Caspase-8, which are not sufficient to trigger TRAIL-induced apoptosis. Reconstitution of Caspase-8 expression is sufficient to sensitize back these cells to TRAIL (Zhang et al, 2005). We observed that DLD1/TRAIL-R cells are also resistant to Fas-induced apotosis (Figure 7F). We reconstituted these cells with Caspase-8-wt or Caspase-8-Y380F (Figure 7E). Immunoblotting with our phospho-specific antibody revealed that Caspase-8-wt but not its unphosphorylatable counterpart is phosphorylated on Tyr380 in these cells, suggesting the presence of endogenous active Src (Figure 7E). Interestingly, reconstitution of Caspase-8 expression sensitize back these cells to Fas-induced apoptosis. However, cells reconstituted with Caspase-8-wt, phosphorylatable on Tyr380, are significantly less sensitive to Fas than cells reconstituted with the unphosphorylatable mutant, Caspase-8-Y380F (Figure 7F), supporting the idea that tyrosine phosphorylation impairs Caspase-8 activity in colon cancer.

Discussion

We are investigating the crosstalk between non-receptor tyrosine kinases and caspases and whether these two families of proteins can modulate each other's function through their enzymatic activities. Surprisingly, however, despite the fact that tyrosine kinases play a fundamental role in cell survival and growth, no caspases have been identified as substrates of tyrosine kinases. Nevertheless, inhibition of tyrosine kinases triggers apoptosis in many experimental systems (Griffiths et al, 2004) and the tyrosine phosphatase SHP-1 sensitizes cells to Fas-induced cell death (Su et al, 1995; Daigle et al, 2002).

Caspase-8 is absolutely required for Fas-induced apoptosis (Juo et al, 1998). Therefore, we tested whether non-receptor tyrosine kinases impinge on Fas-signaling directly targeting Caspase-8. Here we show that Src kinase directly phosphorylates Caspase-8. Src activity triggers endogenous Caspase-8 tyrosine phosphorylation and protects cells from Fas-induced apoptosis, suggesting that tyrosine phosphorylation directly modulates Caspase-8 activity and function. Using an enzymatic assay for Caspase-8 activity, we could show that the expression of a constitutively active Src severely impairs endogenous Caspase-8 activation, similarly to what was observed upon expression of a potent inhibitor of caspases such as CrmA. Moreover, the expression of a constitutively active Src delays Procaspase-8 processing and the following accumulation of its cleavage products. These events normally occur upon Fas-stimulation as the final result of Procaspase-8 recruitment to the DISC, dimerization and activation. Since Fas-induced apoptosis is strictly dependent on Caspase-8 activation, and Caspase-8 activation upon Fas-stimulation ends with its autoprocessing, the observation that Src kinase activity downregulates Caspase-8 cleavage, suggests that it directly impinge on Caspase-8 activation.

Mass spectrometry and point mutagenesis on immunoprecipitated Caspase-8 from cells cotransfected with Caspase-8 and an active Src identified a single phosphorylation on Tyr380. Interestingly, a database search using ScanSite (Songyang et al, 1995) identified this site as an optimal putative phosphorylation site for Fgr kinase, a member of the Src family kinases.

The generation of Caspase-8-Y380F mutant that cannot be phosphorylated by Src kinase allowed us to test the significance of this phosphorylation on Caspase-8 activity and function. Remarkably, Tyr380Phe mutation per se does not affect the function of Caspase-8, neither in S. pombe nor in the reconstituted Caspase-8-deficient mammalian cells. However, Src kinase expression relieves yeast from Caspase-8 but not from Caspase-8-Y380F toxicity, indicating that Tyr 380 phosphorylation modulates Caspase-8 activity. Moreover, the experiments on the reconstituted Caspase-8-deficient mammalian cells show that Tyr 380 phosphorylation modulates Fas-induced apoptosis.

To further investigate the biological relevance of this phosphorylation, we asked whether Caspase-8 might be tyrosine phosphorylated in any physiological or pathological context where Src kinase activity is upregulated. We observed that the activation of endogenous Src kinase activity upon EGF receptor stimulation (Bromann et al, 2004) triggers Caspase-8 tyrosine phosphorylation. Moreover, Src kinase activity is necessary for the antiapoptotic effect of EGF on Fas-induced cell death. This finding, together with the data obtained in yeast and in Caspase-8-deficient mammalian cells, allows speculating that EGF protection relies on Src's ability to phosphorylate and modulate Caspase-8 activity.

Src family kinases are upregulated in epithelial and non-epithelial tumors (Frame, 2002; Summy and Gallick, 2003; Warmuth et al, 2003). Significantly, we were able to detect tyrosine phosphorylated Caspase-8 in 80% of human colon tumors. This event matches the observation that Src kinase activity is also upregulated in the same percentage of these tumors (Allgayer et al, 2002). Moreover, we could show that Caspase-8 is specifically phosphorylated on Tyr380 in colon cancer. This finding, together with the data showing that phosphorylation on this residue downregulates Caspase-8 activity, allows us to propose that Src may contribute to cancer formation and development through the downregulation of the Caspase-8-dependent apoptotic machinery. Interestingly, several cancers show genetic alterations that lead to a lack of Caspase-8 activity, supporting the idea that the impairment of Caspase-8 function contributes to cancer development (Teitz et al, 2000; Harada et al, 2002; Kim et al, 2003; Ashley et al, 2005; Soung et al, 2005). Many tumors are resistant to death receptor-induced apoptosis as well as to other treatments that trigger Caspase-8 activation (Nicholson, 2000; Igney and Krammer, 2002), and carcinoma cells can be sensitized to TRAIL by treatment with chemotherapeutic drugs that enhance Caspase-8 activation (Ganten et al, 2004). Interestingly, inhibition of Src sensitizes human colon cancer cell lines to Fas-induced apoptosis (Griffiths et al, 2004). Importantly, we could show that Caspase-8 phosphorylation on Tyr380 contributes to Fas resistance in colon cancer cells. These observations suggest that Caspase-8 activity plays an important role also in cancer resistance to therapy.

Tyr380 is conserved through evolution in other mammals, such as chimp and dog, but not in rat and mouse. However, it is conserved also in the putative linker region of other species such as chicken, zebrafish and Xenopus. This observation suggests that Src may not trigger Caspase-8 tyrosine phosphorylation in rodents and that this circuit may not be functional in mice and rats. Importantly, despite the overall similarities between humans and mice transformation and carcinogenesis, an emerging body of evidence indicates that there are fundamental differences in how the process of tumorigenesis occurs in the two species (Rangarajan and Weinberg, 2003). Overall, humans are more sensitive to epithelial tumors while mice preferentially develop mesenchymal tumors. Moreover, mice are more resistant to the development of colon cancer (Rangarajan and Weinberg, 2003). These differences point to the evolution of species-specific mechanisms that differentially control tumorigenesis, which may account for the absence of Caspase-8 tyrosine phosphorylation in rodents.

Tyr380 lies in the linker region between the large and the small subunits. The p18 and p10 subunits are separated by a linker peptide also in other caspases, such as Caspase-1, 2, 4, 5 and 6 (Cohen, 1997). However, only Caspase-8 shows the presence of a Tyr residue in this region.

No stuctural data are available for Procaspase-8, therefore the conformation and the role of the linker in the control of caspase activation remains speculative. More importantly, we lack any structural information to be able to predict how phosphorylation on Tyr380 modulates Caspase-8 proapoptotic function. Tyrosine phosphorylation may modulate one of the steps of Caspase-8 activation, such as DISC recruitment, dimerization, activation, processing and release of the active tetramer. Our experimental data support the hypothesis that phosphorylation on Tyr380 mainly modulates Caspase-8 activity and function affecting its processing. Phosphorylation on Tyr380 may also drive new protein–protein interactions that either change the conformation of Caspase-8 preventing its activation, or sequester it far from its substrates, impinging on its apoptotic function. Further experiments will address this point. Moreover, it will be interesting to investigate the possibility that this event occurs also in nonapoptotic responses where Caspase-8 activation has been involved, such as lymphocyte differentiation (Su et al, 2005).

Overall, our findings provide a direct link between Src kinase and Caspase-8 activity modulation and suggest that the inhibition of Src kinase activity might be used to sensitize human carcinomas to treatments that exploit death receptor-induced apoptosis.

Materials and methods

DNA constructs

pSGT-SrcY527F, pSGT-SrcY527F/K295M(kin^-), pSGT-Src-SH₃ pRSP-Src, pSGT-Abl-PP, Bcr-Abl and c-Jun were previously described (Barilà et al, 2003). All Src DNAs were generated from chicken Src sequence (c-Src). All human Caspase-8 constructs were derived from pcDNA3-Caspase-8-HA, generously provided from R Beyaert (Ghent University, Belgium). Briefly, pcDNA3-Myc-Caspase-8 was obtained by PCR with specific oligonucleotides designed to insert Caspase-8 sequence into pCDNA3-Myc, digested with BamHI and XbaI, in frame with two Myc tag at the 5'. pEGFP-CrmA was generated from pHD1.2-CrmA (provided by J Yuan, Harvard Medical School, Boston, USA) by PCR amplification with specific oligonucleotides designed to subclone the fragment into pEGFP-C3 (Clontech) using HindIII and ApaI restriction sites. pNU-Caspase-8 was generated by PCR, with specific oligonucleotides designed to subclone Caspase-8 sequence into pNU vector digested with SpeI–BamHI. pCDNA3-Myc-Caspase-8-Y380F and pNU-Caspase-8-Y380F were generated using the QuickChange site-directed mutagenesis kit (Stratagene) using pCDNA3-Myc-Caspase-8 and pNU-Caspase-8, respectively, as templates. pEGFP-N1-Spectrin was kindly provided by A Beavis and R Kalejta (Princeton University, USA).

Antibodies and other reagents

The following antibodies and reagents were used: anti-Caspase-8 (clone 5F7, MBL), anti-phosphotyrosine (clone 4G10, UBI), anti-Src monoclonal antibodies 2–17 (Microbiological Associates), phospho-Src-Y416 (Cell Signaling), EGF human recombinant (UBI), anti-Fas IgM monoclonal antibody (CH11; UBI), Hoechst 33342 (Molecular Probes) A mouse monoclonal phosphospecific antibody, p-Casp-8-(Y380), was generated (EMBL, Mouse Monoclonal Antibodies Facility) against a Caspase-8 phosphopeptide, CGEQ^*YLEMDLSSPQTR, where ^* represents the phosphotyrosine (Sigmagenosys).

Cell culture and transfections

HEK293 and HeLa cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and transfected with the calcium phosphate precipitation method as previously described (Barilà et al, 2003). Wild-type Jurkat cells and Caspase-8-deficient Jurkat mutant, I9.2 (ATCC), were cultured in RPMI 1640 medium with 10 mM HEPES, 1.0 mM sodium pyruvate, 10% fetal bovine serum. Jurkat I9.2 were transiently transfected using Lipofectamine 2000 (Life Technologies) essentially following the manufacturer's intruction. Briefly, 1 10⁶ cells were transfected in 1 ml with 6 l LipofectAMINE 2000 and 4 g of total DNA (0.5 g pEGFP-N1-Spectrin, 1 g pcDNA Caspase-8-wt, 1 g pcDNA Caspase-8-Y380F, 2.5 g pSGT Src Y527F), and stimulated to undergo apoptosis with 500 ng/ml of anti-Fas mAb. DLD-1/TRAIL-R, kindly provided by B Fang (University of Texas, USA), were grown in DMEM, supplemented with 10% fetal bovine serum, transfected as described for Jurkat cells and stimulated to undergo apoptosis with 500 ng/ml of anti-Fas mAb plus 1 g/ml cycloheximide for 3 h.

Analysis of apoptosis

Transfected or nontransfected HeLa cells were serum-deprived for 24 h. Cells were then treated with 250–500 ng/ml of anti-Fas mAb plus 1 g/ml cycloheximide, in the presence or absence of 100 ng/ml EGF. Apoptosis was determined at different times after anti-Fas treatment by counting Hoechst-stained fragmented nuclei among GFP-positive cells. Blind counting of at least 200 cells were performed for each group.

Fas-stimulated Jurkat and DLD1/TRAIL-R cells were analyzed for DNA fragmentation by FACS analysis. Apoptosis was quantitated by propidium iodide nuclear staining within the electronically gated GFP-positive population, using a FACScan (Becton Dickinson).

Specific apoptosis was determined as follows: (% of apoptotic cells with anti-Fas-% of apoptotic cells without anti-Fas)/(100-% of apoptotic cells without anti-Fas).

Immunoblotting and immunoprecipitation

Cell extracts were prepared in IP buffer (50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 1% NP-40, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 25 mM NaF, 1 mM orthovanadate, 10 g/ml TPCK, 5 g/ml TLCK, 1 g/ml leupeptin, 10 g/ml soybean trypsin inhibitor, 1 g/ml aprotinin) (Barilà et al, 2003). For immunoblotting, 50–100 g of protein extract were separated by SDS–PAGE, blotted onto nitrocellulose membrane and detected with specific antibodies. For immunoprecipitation, protein extracts prepared as above were incubated for 1–2 h with specific antibodies previously conjugated to proteinA-sepharose (Amersham). Immunocomplex were then resolved and analysed by SDS–PAGE. All immunoblots were revealed by ECL (Amersham).

Mass spectrometric analysis (MS)

Bands corresponding to Caspase-8 were excised from the gel, subsequently reduced, alkylated and digested overnight with bovin trypsin. Proteins were unambiguously identified by MALDI-TOF peptide mass mapping. 1 l of the supernatant of the digestion was loaded onto the Maldi target using the dried droplet technique and -cyano-4-hydroxycinnamic acid (HCCA) as matrix. Maldi-MS measurements were performed on a Voyager-DE STR (Applied Biosystems) time of flight (TOF) mass spectrometer and processed via the Data Explorer software. Phosphorylated peptides were analyzed by Tandem MS experiments performed on a Q-Star pulsar (QqTof hybrid system from PE SCIEX Instrument, Toronto, Canada).

Phosphopeptides were purified with Immobilized Metal Affinity Chromatography (IMAC). IMAC was performed using the Phosphopeptide Isolation Kit (Pierce Biotechnology) according to the manufacturer's instructions. Phosphopeptides, detected by MALDI-TOF MS, were confirmed by alkaline phosphatase treatment on target.

Yeast strains and culture conditions

All yeast studies employed the S. pombe strain SP200 (h^-sleu1-32 ura4 ade2-10). Growth conditions, media and induction were according to Superti-Furga et al (1993). Cells were grown either in full medium with addition of adenine (YEA) or minimal medium containing adenine (PMA) and leucine or uracil where appropriate. To repress the nmt1 promoter, cells were kept in medium containing 4 M thiamine. Transformation was carried out by the lithium acetate (LiAc) method as described. Protein extracts from S. pombe cells were produced as described previously (Superti-Furga et al, 1993).

Caspase-8 activity assays

To determine Caspase-8 activity in HeLa extracts, 24 h after transfection, cells were induced to undergo apoptosis with 250 ng/ml of anti-Fas mAb plus 1 g/ml cycloheximide. Proteins were extracted from apoptotic cells in protein IP buffer. Ac-IETD-pNA colorimetric assay was performed at 37°C in 96-well plate, in 100 l assay buffer (50 mM Hepes pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 nM DTT) containing 100 g protein extract. Reaction was started by the addition of 200 M Ac-IETD-pNA and monitored by reading the absorbance at 405 nm. Absorbance over the linear portion of the curve was converted in nmol of substrate hydrolyzed/min using an extinction coefficient for p-nitroaniline of 10 500 M-1 cm-1.

Pombe protein extracts were assayed for Caspase-8 activity using Ac-IETD-AMC as a substrate at 37°C in 200 l assay buffer (20 mM Tris, pH 7.4, 0.1 M NaCl, 10% sucrose, 0.1% CHAPS, 10 mM DTT) containing 250 g protein extract. Reaction was started by the addition of 10 M Ac-IETD-AMC. Cleavage of the substrate as a function of time was monitored reading the absorbance at 460 nm upon excitation at 390 nm.

The enzymatic activity was determined from the linear portion of the curve.

Caspase-8 dimerization assay and BiotinVAD binding

Protein extracts from HeLa cells were incubated for 45 min at 37°C with or without 0.7 M Na citrate to induce Caspase-8 dimerization in the presence of 10 M biotinyl-VAD-fmk, which labels caspases' active sites. Labeled active Procaspase-8 dimers were then immunoprecipitated with agarose-conjugated streptavidin beads and analysed by Western blot.

For Biotin-VAD labeling of apoptotic cell extracts, proteins were extracted in IP buffer and labeled with 10 M biotinyl-VAD-fmk for 45 min at 37°C. Labeled proteins were then immunoprecipitated with agarose-conjugated streptavidin beads and analysed by Western blot.

Analysis of Caspase-8 tyrosine phosphorylation in human colon cancer

Colorectal adenocarcinoma and corresponding normal tissue were obtained from 23 patients who underwent surgical resection. Each sample was rapidly frozen in liquid nitrogen and homogenized on ice, in lysis buffer (IP buffer plus 0.1%Triton X-100).

Statistical methods

All data were analyzed and presented as means.d. (n<10). The significance of differences between populations of data were assessed according to the Student's two-tailed t-test with a level of significance of at least P<0.05 (alpha conventionally equal to 0.05). This analysis arises in the problem of estimating the mean of a normally distributed population when the sample size is small.

Supplementary data

Supplementary data are available at The EMBO Journal Online.

Acknowledgements

We acknowledge R Beyaert, P Vandenabeele, J Yuan, A Beavis, R Kalejta, B Fang, D Nicholson, C Pop and G Salvesen for kindly providing reagents and suggestions, O Hantschel and MG di Bari for helpful discussion and critical reading of the manuscript, D Serio for technical assistance and N Ventura for helping with the FACs analysis. DB is an Assistant Telethon Scientist and is supported by the Italian Telethon Grant (TCP00061), SC has been supported by the Italian Telethon Foundation, VS and AR have been supported by the Italian Foudation for Cancer Research (FIRC). This work has been supported by grants from the Italian Telethon Foundation (TCP00061), from the Italian Association for Cancer Research (AIRC) and from the Italian Compagnia di San Paolo-Imi Bank Foundation to DB. This work was also supported by a grant from AIRC to RT.

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