Calmodulin Binding to the Fas Death Domain

Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca2+-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca2+-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased ∼2-fold early upon Fas activation (at 30 min) and is decreased to ∼50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.

The death receptor, Fas (APO-1/CD95), and its ligand have been identified as important signal mediators of apoptosis (1). Genetic mutations of both Fas and its ligand, FasL, have been associated with lymphoproliferative and autoimmune disorders in mice (2) and humans (3). Furthermore, alterations of Fas expression and/or activity have been implicated in uncontrolled expansion of neoplastic cells and induction of apoptosis in T-cells infected with human immunodeficiency virus (4) or expressing the human immunodeficiency virus-1 coat glycoprotein, gp160 (5).
The structural organization of Fas indicates that this receptor is a member of the tumor necrosis factor receptor (TNFR) 1 superfamily, which also includes other death receptors, such as the tumor necrosis factor-related apoptosis-inducing ligand receptor-1 (TRAIL-R1/DR4) and TRAIL-R2/DR5. These receptors have a cysteine-rich extracellular domain, which is a common feature of the TNFR superfamily, and a homologous cytoplasmic sequence termed the death domain (DD) (6). The Fas DD consists of six amphipathic ␣-helices arranged antiparallel to one another, with side chains of hydrophobic residues forming an extensive network of interactions that constitute the core of the protein. Helices 1 and 2 are centrally located with helices 3 and 4 on one side and helices 5 and 6 on the other (7). Fas-FasL ligation leads to a clustering of the DDs of the receptors (7), and an adapter protein, Fas-associated death domain-containing protein (FADD) (8), then binds through its own DD to the clustered-receptor DD. FADD also has a death effector domain that binds to an analogous domain in caspase-8 (9). Several other cytoplasmic proteins that can bind to Fas have been identified, including Daxx (10), FAP-1 (Fas-associated phosphatase-1) (11), RIP (receptor-interacting protein) (12), and FAF1 (Fas-associated factor 1) (13).
Calmodulin (CaM) acts as a major Ca 2ϩ sensor and regulator through its interaction with a diverse group of cellular proteins. There is 100% identity in the amino acid sequence of CaM among vertebrates, with multiple genes encoding identical CaMs (14). Structurally, CaM is a dumbbell shape formed by two globular domains at its C and N termini that are connected by a flexible helical linker region. The globular ends each contain a pair of Ca 2ϩ -binding motifs called EF-hands, and the binding of Ca 2ϩ to CaM exposes a hydrophobic surface that is responsible for binding to various target proteins (15). The function of CaM is not confined to its Ca 2ϩ -bound form, because Ca 2ϩ -free CaM (apocalmodulin) can recognize different target proteins (16). More than 30 CaM-binding proteins have been identified, including enzymes such as kinases, phosphatases, and nitric-oxide synthase, as well as receptors, ion channels, G-proteins, and transcription factors (17)(18)(19). Many known Ca 2ϩ -dependent CaM-binding proteins possess a region that is characterized by an amphipathic helix consisting of ϳ20 amino acid residues. This region contains positively charged amino acids interspersed among hydrophobic residues (20). Specific characteristics of the region for several CaM-binding proteins include a net positive charge, moderate hydrophilicity, and a moderate to high helical hydrophobic moment (21). However, these criteria do not always identify the CaM-binding region in target proteins. From sequence comparisons of CaMbinding peptides and CaM-binding domains within the CaMregulated proteins, three classes of CaM-binding motifs have emerged. These classes include a modified variant of the IQ motif as a consensus for Ca 2ϩ -independent binding and two related motifs for Ca 2ϩ -dependent binding, termed 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) and 1-5-10 (XXX-(FILVW)XXX(FILV)XXXX(FILVW)) based on the conserved hydrophobic residues within these motifs (14).
Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site in the DD of Fas. We also show changes in the Fas-CaM interaction during Fas-mediated apoptosis. These results suggest that CaM is a novel regulator of Fas-mediated apoptosis.

EXPERIMENTAL PROCEDURES
Cells, Antibodies, and Reagents-Jurkat cells were purchased from ATCC (Manassas, VA). Calmodulin-Sepharose® 4B and GST antibody were from Amersham Biosciences, and Sepharose CL-4B was from Sigma. Antibodies were purchased from the following companies: antibodies for Fas (monoclonal, B-10) and TNFR from Santa Cruz Biotechnology (Santa Cruz, CA), DR4 antibody and agonistic Fas antibody (clone CH11) from Upstate Biotechnology (Lake Placid, NY), caspase-8 antibody from Cell Signaling Technology (Beverly, MA), and DR5 antibody from Sigma. Purified CaM (from bovine brain) was obtained from Calbiochem (La Jolla, CA). The monoclonal antibody to CaM was developed as described previously (22).
CaM Pull-down Assay and Immunoprecipitation-Cell lysates in lysis buffer (phosphate-buffered saline containing 1% Triton X-100) or purified GST fusion protein diluted in binding buffer (50 mM Tris (pH 7.6), 120 mM NaCl, 1% Brij) were incubated with Calmodulin-Sepha-rose® 4B or control Sepharose for 1-2 h at 4°C as indicated in the figure legends. After washing, beads were boiled in SDS sample buffer and separated by SDS-PAGE followed by Western blotting. Immunoprecipitation with CaM antibody was performed using the Seize™ primary mammalian immunoprecipitation kit (Pierce) according to the manufacturer's directions.
Western Blotting-Proteins were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). For Western blot for CaM, membranes were then fixed in 0.2% glutaraldehyde in Tris-buffered saline for 30 min. The membranes were blocked in 2% nonfat milk and incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Blots were developed using enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences).
Generating Fas Fragments by Polymerase Chain Reaction-Various fragments of human Fas were generated by PCR with the following forward (F) and reverse (R) primers containing EcoRI (italics) and XhoI Site-directed Mutagenesis-The QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to make Fas point mutations V254N, T241F, G247F, and F248A. Primers for the mutagenesis were purchased from Invitrogen.
Preparation of GST Fusion Protein-Wild-type or mutated Fas cytoplasmic regions were inserted into the pGEX5-1 vector using EcoRI and XhoI sites. The expression and purification of GST fusion proteins were performed according to the manufacturer's directions for GST expression and purification systems (Amersham Biosciences).
Computer-based Modeling of the CaM-Fas Interaction-The model of the interaction between CaM and the Fas DD helix 2 was prepared using RIBBONS (23). This model was based on the crystal structure of calmodulin and calmodulin-dependent protein kinase II peptide complex (24).

Binding of Fas to CaM-Sepharose-The interaction between
Fas and CaM was first demonstrated by a CaM pull-down assay. Jurkat cell lysates were incubated with calmodulin-conjugated-Sepharose (CaM-Sepharose) or control Sepharose and Western blotted with Fas antibody. The results show that Fas is detected in CaM-Sepharose-bound proteins but not in control Sepharose-bound proteins (Fig. 1A). We also used lysates of other cells for the CaM pull-down assay: the normal human mammary epithelial cell line (MCF-10a), the human breast cancer cell line (MCF-7), the human pancreatic cancer cell lines (MIAPaca-2, PANC-1), the human cholangiocarcinoma cell line (Sk-ChA-1), primary cultures of mouse osteoclasts, and a mouse macrophage-lineage cell line (RAW 264. 7). From all cell lysates tested, Fas was detected in CaM-Sepharose-bound proteins (data not shown).
Because Fas belongs to the TNFR superfamily in which many members have structural similarity, we were interested in whether other receptors in the TNFR family interact with CaM. Western blot analysis of CaM-Sepharose-bound proteins showed no interaction between TNFR-1 (p55), DR4, or DR5 and CaM (Fig. 1A).
Many of the CaM-target protein interactions are Ca 2ϩ -dependent (14). To investigate the Ca 2ϩ dependence of Fas binding to CaM-Sepharose, the CaM pull-down assay was performed with cell lysates in Ca 2ϩ -supplemented buffer (1 mM) or EGTA-containing buffer (1 mM). Fig. 1B shows that the presence of EGTA abolished the binding of Fas to CaM-Sepharose, indicating that Fas binding to CaM-Sepharose is Ca 2ϩ -dependent.
Co-immunoprecipitation of CaM and Fas-Because Fas binding to CaM-Sepharose was observed, we next investigated whether endogenous CaM and Fas interact in vivo. We immunoprecipitated CaM from the lysates of Jurkat cells with CaM antibody and tested for associated Fas by Western blot analysis. As shown in Fig. 2, Fas was co-immunoprecipitated with CaM demonstrating that the Fas-CaM complex is present in cells. Direct Interaction of CaM and GST-Fas-Although we observed a Fas-CaM association in vitro and in vivo, we could not exclude the possibility that their association is mediated by other Fas-binding proteins present in the cell lysates. To demonstrate the direct binding between Fas and CaM, we performed in vitro binding assays. The Fas cytoplasmic domain (amino acids 191-335) was expressed in Escherichia coli as a GST fusion protein affinity-purified on glutathione-Sepharose. This GST-Fas protein was clearly detected by the Fas antibody, which has an epitope in the cytoplasmic domain of Fas, confirming that the Fas cytoplasmic domain is attached to GST. We incubated GST-Fas with CaM-Sepharose in a binding buffer containing Ca 2ϩ and tested for the interaction of GST-Fas to CaM-Sepharose by Western blot using a GST antibody. As shown in Fig. 3A, GST-Fas bound to CaM-Sepharose but not to control Sepharose. GST alone did not show any interaction with CaM-Sepharose.
We also tested for the binding of purified CaM to GST-Fas. After the incubation of GST-Fas or GST protein alone with purified CaM, GST-associated proteins were pulled down with glutathione-Sepharose, and the presence of CaM was determined by Western blot using the CaM antibody. Fig. 3B shows that CaM was pulled down by GST-Fas. These results confirm the direct interaction between Fas and CaM.
Mapping of the Site on Fas Involved in Binding to CaM-To determine the region in the cytoplasmic domain of Fas that is required for binding to CaM, we prepared a series of Fas deletion mutants that were expressed as GST fusion proteins (Fig. 4A) and purified on glutathione-Sepharose. These Fas deletion mutants were incubated with CaM-Sepharose, and the amount of CaM-bound Fas was determined by Western blot using a GST antibody. As shown in Fig. 4B, fragments containing amino acids 191-335 (the entire Fas cytoplasmic domain; numbers represent amino acid sequence numbers), 191-320, and 191-270 bound to CaM-Sepharose; however, the 191-229 amino acid fragment did not bind to CaM-Sepharose suggesting that the 230 -270 fragment contains the CaM-binding site(s). To further confirm that the 230 -270 fragment contains the CaM-binding site(s), we generated 230 -270 and 271-335 Fas fragments as GST fusion proteins. A CaM pull-down assay revealed that the 230 -270 fragment, but not the 271-335 fragment, binds to CaM (Fig. 4B).
The CaM-binding ability of Fas amino acids from 230 through 270 was further confirmed by incubation with pure CaM. Three different GST fusion Fas cytoplasmic regions, 191-229, 230 -270, and 271-335, were incubated with purified CaM and pulled down with glutathione-Sepharose. Western blot analysis, using the CaM antibody, demonstrated that only the 230 -270 fragment pulls down CaM (Fig. 4C). We also incu-bated the GST-Fas-(230 -270) fragment with CaM-Sepharose in the presence of EGTA and confirmed that the interaction of the 230 -270 fragment with CaM is Ca 2ϩ -dependent (Fig. 4D).
Prediction of CaM-binding Motif-Fas amino acids 230 -270 cover the helices 1, 2, and 3 of the DD of Fas. We examined the amino acid sequence of 230 -270 to identify the possible CaMbinding site. The 41 amino acids between amino acid residue 230 and 270 were checked for CaM-binding regions using a Web-based data base, the cellular calcium information server, 2 provided by Dr. Ikura's laboratory at the Ontario Cancer Institute (see also Ref. 19). This sequence analysis normalizes scores (0 -9) based on multiple criteria, e.g. hydropathy, ␣-helical propensity, residue weight, residue charge, hydrophobic residue content, helical class, and the occurrence of particular residues. A repeated high score indicates the location of a putative binding site (25). The search results showed a series of high scores (ϳ9) for helix 2 and the connected loops (Fig. 5).
Although CaM binds to diverse sequences in a diverse group of proteins, sequence comparisons of CaM-binding peptides and CaM-binding domains within CaM-regulated proteins have classified three recognition motifs for CaM interaction (14). They include a modified variant of the IQ motif as a consensus for Ca 2ϩ -independent binding and two related motifs for Ca 2ϩdependent binding, termed 1-8-14 ((FILVW)XXXXXX(FAIL-VW)XXXXX(FILVW)) and 1-5-10 (XXX(FILVW)XXX(FILV)-XXXX(FILVW)) based on the conserved hydrophobic residues within these motifs. In the Fas 230 -270 region, we predicted a possible CaM-binding motif, LSQVKGFVRKNGV (Fig. 5, underlined). This sequence fits the 1-5-10 motif (XXX(FIL-VW)XXX(FILV)XXXX(FILVW)) with a net charge of ϩ3. This region corresponds to helix 2 and the loop between helices 2 2 The data base is available at calcium.uhnres.utoronto.ca.

FIG. 3. Interaction of GST-Fas and CaM. A, the whole Fas cytoplasmic domain as a GST fusion protein (GST-Fas) or GST alone was incubated with CaM-Sepharose (CaM-S) or control Sepharose (S) in
Ca 2ϩ -containing (1 mM CaCl 2 ) binding buffer for 2 h at 4°C. The beads were collected and Western blotted with GST antibody. Input, onethirtieth of the amount of protein used for incubation with beads. B, GST or GST-Fas was incubated with purified CaM for 2 h, followed by an additional 1-h incubation with glutathione-Sepharose. Beads were collected and Western blotted (WB) with CaM or GST antibody. and 3 of the Fas DD. Although a part of helix 1 was also highly scored by the cellular calcium information server data base, 2 we could not find a classical CaM-binding motif in this region; it does not contain charged amino acids. Taken together, these results predict that the helix 2 area, containing the 1-5-10 motif, is the most likely CaM-binding site.
The Effect of Fas Point Mutations on CaM Binding-Next, we changed single amino acids in the putative CaM-binding site to determine whether these changes affect Fas-CaM binding. First, we changed valine 254 to asparagine (V254N). Valine 254 is located in the loop between helices 2 and 3 and is one of the hydrophobic residues that meet the criteria for the 1-5-10 motif. Interestingly, V254N is analogous to an identified mutant allele of Fas in the cg strain of lpr-autoimmune mice that have been shown to be deficient in Fas-mediated apoptosis. In addition, this mutation has been shown to reduce both the self-association of the Fas DD and binding to FADD, suggesting that it alters the protein structure (7). To determine CaMbinding to this mutation, we generated the whole cytoplasmic region of Fas with the V254N mutation as a GST fusion protein and tested for binding of this mutated Fas fragment to CaM-Sepharose. As shown in Fig. 6A, the V254N mutant has a reduced CaM-binding ability when compared with wild-type Fas.
To predict other amino acid changes that might possibly affect CaM-Fas binding, we modeled the three-dimensional structure of CaM bound to a peptide corresponding to the Fas 230 -270 fragment (Fig. 6B). A threonine 241 mutation to phenylalanine (T241F), a glycine 247 mutation to phenylalanine (G247F), and a phenylalanine 248 mutation to alanine (F248A) are predicted to be substitutions that will maximally affect binding to CaM, because three-dimensional computer modeling predicts that these residues have close interactions with CaM (Fig. 6B).
The effect of these mutations on CaM binding was tested using CaM-Sepharose. Whole Fas cytoplasmic regions containing T241F, G247F, or F248A were prepared as GST fusion proteins and incubated with CaM-Sepharose, and the amount of CaM-bound Fas was determined by Western blot with a GST antibody. Fig. 6C shows that all three mutations have reduced CaM-binding ability when compared with wild type. These results support our prediction that helix 2 of the Fas DD is responsible for the interaction with CaM.
Changes in Fas-CaM Interaction during Fas-mediated Apoptosis-Because CaM binds to the Fas DD and may therefore regulate Fas-mediated apoptosis, we tested whether the Fas-CaM interaction is altered during the early stage of Fas-mediated apoptosis. We treated Jurkat cells with the agonistic Fas antibody (clone CH11) for 30 min, 1 h, and 2 h. Cell lysates were immunoprecipitated with CaM antibody, and the amount of Fas bound to CaM was assessed by Western blot for Fas. Interestingly, upon the activation of Fas with the agonistic antibody, the binding between Fas and CaM was increased by ϳ2-fold in 30 min. This binding was then decreased to ϳ50% of The amino acid sequence from 230 through 270 in the Fas cytoplasmic domain was analyzed to predict CaM-binding sites, using a CaM target data base. The numbers under the sequences indicate normalized scores (0 -9) based on the evaluation criteria for CaMbinding sites (see "Results" for details). The predicted 1-5-10 CaMbinding motif is underlined. Arrows with solid lines indicate hydrophobic residues, and arrows with dotted lines indicate positively charged residues. Coils above the sequence represent ␣-helical regions. the control after 2 h (Fig. 7). These results indicate that CaM binds to Fas with more affinity in response to Fas activation followed by its release from Fas, suggesting that Fas-CaM interaction is functionally significant.

DISCUSSION
Fas is one of the well characterized death receptors that initiates the extrinsic pathway of apoptosis. After Fas was first discovered as a mediator of cell death (1), considerable effort was focused on identifying the downstream molecules, especially those that bind to the Fas cytoplasmic domain. Although several Fas-binding molecules such as FADD (8), Daxx (10), FAP-1 (11), RIP (12), and FAF1 (13) have been identified, many aspects of Fas-mediated apoptosis remain undetermined. Moreover, Fas can not only induce apoptosis in many cell types, but it can also mediate cell proliferation in certain cell types (26,27), suggesting that the engagement of Fas and its ligand transduces a variety of biological effects, which may be mediated by interactions of the Fas cytoplasmic domain with various molecules.
In this report, we demonstrated for the first time that CaM interacts with the Fas DD directly and that the interaction between CaM and Fas is altered during Fas-mediated apoptosis, suggesting a functional significance of this interaction. We confirmed CaM-Fas binding by several methods, including a CaM pull-down assay with CaM-Sepharose, immunoprecipitation, and an in vitro binding assay with purified proteins. Additionally, we identified a CaM-binding site in the Fas DD by analyzing the Fas amino acid sequence and using deletions and point mutations of Fas.
These findings are important not only for understanding Fas-mediated apoptosis but also for expanding the knowledge of CaM, which has been known to interact with diverse molecules. Although CaM interacts with a large number of proteins via diverse binding motifs, even more interesting is the diversity of the molecular and functional roles of CaM binding. The interaction of CaM with CaM-dependent serine/threonine protein kinases (CaM kinase I/II/IV, CaM kinase kinase, and myosin light chain kinase) and the phosphatase calcineurin displaces autoinhibitory domains in the proteins to induce full activation of these enzymes. CaM binding also causes rearrangement of key switches to create the active site in target proteins (28,29) and dimerization of membrane proteins, such as the small conductance Ca 2ϩ -activated potassium channels (29,30).
Based on such diverse target-protein activation mechanisms, it is of interest to consider the role of CaM that is bound to Fas. We observed that a C-terminal 15-amino acid deletion mutant of Fas (191-320) strongly interacts with CaM, relative to the association of the whole Fas-cytoplasmic domain with CaM (Fig. 4B). Interestingly, it has been demonstrated that the interaction between Fas and FADD is also enhanced when the C-terminal 15 amino acids of Fas are deleted (8). This deletion probably causes a conformational change in Fas, which helps both FADD and CaM gain access and bind to Fas. Therefore, it is possible that CaM may compete with FADD for binding to Fas. It is also possible that CaM acts as a bridging molecule between different proteins, because CaM-binding sites may not always exist on a single target protein (29). We screened the CaM-binding abilities of several molecules, which are known as DISC (death-inducing signaling complex) components, and identified one DISC component, c-FLIP, as a CaM-binding molecule, 3 consistent with our hypothesis that CaM acts as a bridging molecule between the Fas DD and other DISC components.
In this report, we have shown that during Fas-mediated apoptosis the interaction between Fas and CaM is altered, and CaM binding to Fas was increased in 30 min after Fas activation and then decreased after 2 h (Fig. 7). Together with our published and unpublished data showing that CaM antagonists can induce apoptosis in human cholangiocarcinoma cells (31,32) and Jurkat cells, 3 the decrease in Fas-CaM binding 2 h after Fas activation suggests that the release of CaM from Fas facilitates apoptotic pathways. However, the reason for the initial increase in the binding of CaM to Fas 30 min after Fas activation is less obvious. Interestingly, this biphasic Fas-CaM interaction is somewhat analogous to the pattern of ERK1/2 activation in response to Fas activation. It has been reported that the activation of ERK1/2 is induced rapidly (in ϳ5 min) after agonistic Fas antibody treatment and then is down-regulated after 1 h (33). In addition, ERK1/2 activation has been shown to inhibit Fas-mediated apoptosis through suppression of cleavage caspase-8 and Bid, and pretreatment with an inhibitor of the ERK1/2 activation pathway sensitizes cells to Fas-mediated apoptosis (34). Similarly, we have observed that caspase-8 cleavage was accelerated when cells were pretreated with CaM antagonists before Fas activation. 3 Therefore, it is possible that the initial increase in Fas-CaM binding plays an inhibitory role as a "counteraction" or a "buffering action" to delay or suppress the early steps of Fas-mediated apoptosis, including caspase-8 cleavage. The exact mechanism by which CaM affects Fas-mediated apoptosis is under investigation in our laboratory.
Although we demonstrated that CaM binding to Fas requires Ca 2ϩ molecule (Figs. 1B and 4D), Ca 2ϩ may not be the sole regulator of CaM-Fas binding in vivo. Changes in intracellular Ca 2ϩ concentration during Fas-mediated apoptosis have been documented by several groups. Sen et al. (35) measured intracellular Ca 2ϩ concentration every 15 min during the first 2 h of Fas-mediated apoptosis in Jurkat cells. They observed no change in intracellular Ca 2ϩ concentration until 60 min after Fas activation and a remarkable increase after 2 h. Scoltock et al. (36) also reported that agonistic Fas antibody treatment of Jurkat cells resulted in a sustained increase in intracellular Ca 2ϩ commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. We have also observed an increase in intracellular Ca 2ϩ concentration at 4 and 8 h after Fas activation in carcinoma cells (32). Nevertheless, we observed a deceased CaM-Fas binding 2 h after Fas activation (Fig. 7). Given this information, we propose that the dynamic changes in Fas-CaM interaction during the early stage of Fas-mediated apoptosis may not correlate simply with overall intracellular Ca 2ϩ concentration. Although Ca 2ϩ is necessary for Fas-CaM interaction, it is likely that other factors, such as conformational changes of the Fas cytoplasmic domain after Fas activation and recruitment of other molecules to Fas, also regulate the ability of CaM to bind to the Fas DD.
It is still possible that the alteration of Fas-CaM binding is affected, at least partially, by the local changes of intracellular Ca 2ϩ concentration. Generally, Ca 2ϩ -binding dyes have been used to measure the intracellular Ca 2ϩ concentration, and the results represent the average Ca 2ϩ concentration in the cyto-plasm. However, Ca 2ϩ is not evenly distributed in the cytoplasm; the Ca 2ϩ concentration may be much higher within the microdomains around the mouths of Ca 2ϩ channels in the plasma membrane or on the internal Ca 2ϩ stores (37). Therefore, the localized Ca 2ϩ concentration near the Fas cytoplasmic domain might regulate CaM binding, which could be proven by more detailed imaging of Ca 2ϩ compartmentalization.
The observations in this report provide novel insights into both elucidation of the mechanism of apoptosis and understanding the function of calmodulin. Fundamental understanding of the role of CaM binding to Fas may also provide a new therapeutic modality with CaM antagonists targeting the Fas-CaM interaction.