Peflin and ALG-2, Members of the Penta-EF-Hand Protein Family, Form a Heterodimer That Dissociates in a Ca2+-dependent Manner*

Yasuyuki Kitaura, Shinji Matsumoto, Hirokazu Satoh, Kiyotaka Hitomi, and Masatoshi MakiDagger

From the Laboratory of Molecular and Cellular Regulation, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Received for publication, September 21, 2000, and in revised form, February 1, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peflin, a newly identified 30-kDa Ca2+-binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca2+. Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca2+ but in the membrane/cytoskeletal fraction in the presence of Ca2+. These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca2+ signaling.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is now accepted that Ca2+ is one of the most versatile second messengers relaying information within cells to regulate their activities such as muscle contraction, secretory events, cell cycle, differentiation, gene expression, and apoptosis. Ca2+ plays its pivotal role through specific classes of Ca2+-binding proteins, most of which possess Ca2+-binding motifs such as endonexin folds, C2 regions, or EF-hands. Many EF-hand type Ca2+-binding proteins have been identified, and they have been classified into dozens of families based on amino acid sequence similarities and number of EF-hand motifs in their molecules (1).

Recently, we classified a new family of proteins possessing domains with five EF-hand-like motifs, and we proposed the name "penta-EF-hand (PEF)"1 as a collective name for these domains (2). The PEF domain was originally found in the Ca2+-binding domain of the small subunit of calpain, an intracellular Ca2+-dependent cysteine protease, by x-ray crystallography (3, 4). Later studies revealed that the PEF domains are present in several other Ca2+-binding proteins such as the calpain large subunit, sorcin (5), grancalcin (6), and apoptosis-linked gene 2 (ALG-2) (7). Whereas sorcin and grancalcin exist as homodimers (8, 9), calpains exist as heterodimers of the large catalytic and small regulatory subunits (10). The bacterially expressed recombinant PEF domain of the calpain small subunit forms a homodimer without the large subunit (11). X-ray crystallographic studies have also revealed that the dimers are formed through a pair of fifth EF-hands (EF-5s) that have lost their Ca2+-binding capacities due to two-residue insertions (3, 4). Therefore, it has been proposed that PEF proteins may form dimers with each other through EF-5, which provides a new interface for the interaction with possible targets.

The calpain small subunit, sorcin, grancalcin, and ALG-2 have hydrophobic domains with variable lengths in the N-terminal regions. In the case of calpains, the hydrophobic N-terminal domains bind to the membranes and play an important role in the change of subcellular localization induced by Ca2+ (12). The N-terminal region of sorcin is required to interact with the membrane-localized annexin VII in a Ca2+-dependent manner (13). Thus, the N-terminal regions of PEF proteins are thought to interact with phospholipids and/or target proteins on membranes.

Previously, we reported a novel PEF protein, peflin (PEF protein with a long N-terminal hydrophobic domain), which was cloned after a homology search for other PEF proteins (14). Peflin is most similar to ALG-2 in the PEF domain and has the longest N-terminal hydrophobic region of proteins in the PEF family. Peflin is expressed in several human cell lines, but its target protein and function have not been determined yet.

In this study, using a monoclonal antibody (MoAb) specific to human peflin, we demonstrated that peflin was co-immunoprecipitated with ALG-2. The peflin/ALG-2 heterodimer dissociated in a Ca2+-dependent manner. The N-terminal hydrophobic domain of peflin was not essential for the heterodimerization. Peflin co-localized with ALG-2 in the cytoplasm and changed the subcellular localization in a Ca2+-dependent manner.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Jurkat cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (0.3 mg/ml), penicillin (100 units/ml) and streptomycin (100 µg/ml) at 37 °C under humidified air containing 5% CO2. Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented as above.

Preparation of Anti-peflin Monoclonal Antibody-- BALB/c female mice were immunized three times with His-tagged N-terminal truncated peflin (His-peflinDelta N) prepared as described previously (14). Hybridomas were generated by polyethylene glycol-mediated fusion of donor splenocytes to the P3 myeloma cell line. Positive hybridomas were identified by enzyme-linked immunosorbent assay and cloned by limited dilution. Cloned hybridomas were transplanted intraperitoneally to BALB/c mice. The IgG fraction was prepared from ascites and purified by the ammonium sulfate precipitation method. Western blotting was performed as described previously (14).

Metabolic Labeling and Immunoprecipitation-- Jurkat cells (1 × 107) were incubated in a 60-mm dish containing 1.5 ml of a methionine/cysteine-free medium for metabolic labeling (Sigma) supplemented with PBS-dialyzed fetal bovine serum to 10% and 35S-labeled amino acid mixture (100 µCi/ml, 70% methionine and 30% cysteine) at 37 °C for 4 h under humidified air containing 5% CO2. Cells were washed with PBS and lysed in buffer A (20 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 0.2% Nonidet P-40, 0.1 mM pefabloc, 25 µM leupeptin, 10 µM E-64, and 1 µM pepstatin) containing 5 mM EGTA or 0.01 mM CaCl2. Aliquots were incubated with indicated antibodies for 4 h at 4 °C and further incubated with protein G-Sepharose 4FF (Amersham Pharmacia Biotech) overnight. Immunocomplexes were washed three times with buffer A and subjected to SDS-PAGE and analyzed by autoradiography using a BAS 2000 system (Fuji Film, Kanagawa, Japan). Anti-human RECK MoAb 32C10A (15) was used as a negative control antibody for immunoprecipitation. Anti-FLAG MoAb M2 was obtained from Stratagene (La Jolla, CA). Anti-mouse ALG-2 polyclonal antibody (PoAb) raised in rabbits was affinity-purified using recombinant human ALG-2 as described previously (16).

Expression Vectors and Transfection-- An EcoRI fragment of the full-length peflin cDNA was inserted into a eukaryotic expression vector, pCXN2 (a derivative of pCAGGS, a kind gift from Dr. J. Miyazaki; Ref. 17), and a BamHI fragment of either a full-length or an N-terminal truncated peflin (peflinDelta N: amino acids 116-284) was inserted in-frame into a pCMV-tag2 vector (Stratagene) for expression as FLAG-tagged protein. A human ALG-2 cDNA was cloned from Jurkat cells by the reverse transcription-polymerase chain reaction method, and a BglII/BamHI fragment was inserted into pCXN2 and pCMV-tag2. One day after HEK293 cells (1 × 106 cells/60-mm dish) had been seeded, the cells were transfected with the expression plasmid DNAs by the conventional calcium phosphate precipitation method. After 48 h, cells were collected and analyzed by the immunoprecipitation and/or Western blotting methods, where aliquots of immunoprecipitated proteins and cell lysates were subjected to SDS-PAGE using comparable amounts of the relevant samples. The DNA transfection efficiency monitored by the expression of a green fluorescent protein construct, pCMV-EGFP (obtained from CLONTECH), was about 20% under a similar condition in separate experiments.

Immunofluorescent Staining-- Cytospin preparations of Jurkat cell suspension (2 × 105 cells/0.2 ml) were prepared by centrifugation using an SC-2 adapter (Tomy Seiko, Tokyo, Japan), fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100/PBS. After blocking with 1% bovine serum albumin in 0.1% Tween 20/PBS, cover glasses were incubated with primary antibodies (anti-peflin MoAb and anti-ALG-2 PoAb) at 4 °C overnight and with secondary antibodies (fluorescein isothiocyanate-conjugated anti-mouse IgG for peflin and rhodamine-conjugated anti-rabbit IgG for ALG-2) at room temperature for 30 min. Immunofluorescences were analyzed by an MRC-1024 Laser Scanning Confocal Imaging System (Bio-Rad).

Subcellular Fractionation-- Subcellular fractionation was performed by lysing cells with a Dounce homogenizer in buffer B (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, and the protease inhibitors as described above), followed by centrifugation at 1,000 × g (4000 rpm by a Sakuma M-150 rotor) for 10 min at 4 °C producing a pellet (see Fig. 7A, P1). The supernatant was further centrifuged at 10,000 × g (13,000 rpm by a Sakuma M-150 rotor) for 10 min at 4 °C producing a second pellet (see Fig. 7A, P2) and then at 100, 000 × g (60,000 rpm by a Beckman TLA 100 rotor) for 30 min at 4 °C producing a third pellet and a supernatant (see Fig. 7A, P3 and S, respectively). Nuclei were purified essentially as described by Dignam and colleagues (18). Briefly, the crude nuclear fraction (P1) was homogenized in buffer B containing 0.1% Triton X-100 and 0.2 M sucrose, layered onto a cushion of buffer A containing M sucrose, and centrifuged at 20,000 × g (18,000 rpm by a Beckman TLS-55 rotor) for 30 min at 4 °C.

Gel Filtration-- Jurkat cells (2 × 108) were washed twice with PBS and lysed in buffer B containing 5 mM EGTA, and the cytosolic fractions (100,000 × g, supernatant) were prepared as above. The cytosolic proteins were fractionated by gel filtration using a Superdex-75 column (1.0 cm × 30 cm; Amersham Pharmacia Biotech). Fractions (0.2 ml each) were collected and analyzed by Western blotting. Recombinant human ALG-2 was prepared essentially as described previously (16) and subjected to gel filtration using the same column.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection of Peflin with Monoclonal Antibody-- Previously, we prepared anti-peflin antiserum and detected a 30-kDa protein as a major band in the lysates of various cell lines (14). The antiserum, however, also cross-reacted with a protein of about 40 kDa, and it remained unknown whether the 30-kDa protein was processed from the 40-kDa protein. In the present study, we prepared a MoAb, named P1G, which was more specific to the peflin protein. As shown in Fig. 1A, the prepared MoAb P1G recognized a 30-kDa protein as a single band in Jurkat cell lysates by Western blotting, whereas the antiserum reacted additionally with other proteins. The MoAb could detect FLAG-tagged peflin (FLAG-peflin) and FLAG-tagged N-terminal truncated peflin (FLAG-peflinDelta N) exogenously expressed in HEK293 cells as differently migrating bands at expected positions. Thus, it was concluded that the 30-kDa protein detected with the antiserum corresponds to an unprocessed peflin molecule. To determine whether MoAb P1G could immunoprecipitate peflin, we performed immunoprecipitation followed by Western blotting. The peflin protein was immunoprecipitated with MoAb P1G (Fig. 1B, lane 3) but not with an irrelevant MoAb 32C10A against human RECK (Fig. 1B, lane 2), which is not expressed in Jurkat cells.



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Fig. 1.   Characterization of peflin MoAb P1G. A, Coomassie Brilliant Blue staining (CBB) and Western blotting (WB) of total Jurkat cell extract (top left) using anti-peflin PoAb (Po) or MoAb (Mo) P1G and Western blotting of FLAG-peflin (lane 1) or FLAG-peflinDelta N (lane 2) expressed in HEK293 cells using MoAb P1G (top right) are shown. Bands corresponding to peflin (30 kDa) and a nonspecifically cross-reacting protein are indicated by an arrow and by an asterisk, respectively, in the top left panel. Schematic structures of peflin proteins are depicted (bottom). B, lysates of Jurkat cells were immunoprecipitated with control MoAb 32C10A or anti-peflin MoAb P1G as described under "Experimental Procedures." Cell lysate (lane 1) and immunoprecipitated proteins (lane 2, 32C10A; lane 3, P1G) were analyzed by Western blotting using P1G. Mouse immunoglobulin heavy and light chains of immunoprecipitated antibodies (IgG-H, IgG-L) of MoAb P1G were also detected by subsequent Western blotting using peroxidase-conjugated anti-mouse IgG as a secondary antibody.

Co-immunoprecipitation of Peflin with ALG-2-- To search for a peflin-interacting protein, we metabolically labeled Jurkat cells with 35S-labeled amino acids, and immunoprecipitated peflin with MoAb P1G. An autoradiogram revealed a protein band of about 22 kDa, which was co-immunoprecipitated with peflin in the presence of the Ca2+ chelator EGTA but not in the presence of CaCl2 (Fig. 2). PEF proteins have the common feature of dimerization with each other (2). For example, sorcin and grancalcin form homodimers (8, 9), and calpains form heterodimers of the large and small subunits (10). Interestingly, heterodimers of the calpain subunits have been reported to dissociate in a Ca2+-dependent manner (19). We thought that peflin might also dimerize with itself or with other PEF proteins. We suspected the co-immunoprecipitated 22-kDa protein to be ALG-2 because it is the 22-kDa protein most similar to peflin in the PEF protein family.



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Fig. 2.   Co-immunoprecipitation of peflin with a 22-kDa protein using MoAb P1G. After Jurkat cells had been labeled with [35S]methionine and [35S]cysteine for 4 h, the cells were lysed in the presence of 5 mM EGTA (E) or 0.01 mM CaCl2 (C) as described under "Experimental Procedures." Whole cell extracts were immunoprecipitated with MoAb 32C10A (control) or MoAb P1G (peflin). Aliquots of immunoprecipitated proteins (Ppt) and cell lysates (Lysate) were subjected to SDS-PAGE using comparable amounts of the relevant samples, autoradiographed by exposing on an imaging plate for 24 h (Ppt) or 1 h (Lysate), and analyzed by a FUJIX Bioimaging Analyzer Station BAS 2000. Asterisks and an arrow indicate nonspecifically precipitated proteins and a 22-kDa protein, respectively. Relative radioactivities of the 30-kDa and 22-kDa bands after subtraction of backgrounds are 556 and 132 photostimulated luminescence units, respectively.

To investigate the interaction of peflin with ALG-2 in Jurkat cells, we performed a combined immunoprecipitation-Western blotting analysis. As shown in Fig. 3, ALG-2 was detected in the immunoprecipitates using anti-peflin MoAb P1G in the presence of EGTA but not in the presence of CaCl2. Anti-ALG-2 PoAb also immunoprecipitated peflin in the presence of EGTA in a complementary experiment. The immunoprecipitation experiments were repeated at least three times under different conditions by varying the concentrations of antibodies and protein G. The figures show representative results. Efficiency of the immunoprecipitation of ALG-2 with anti-ALG-2 PoAb was poor, particularly in the presence of Ca2+ (see "Discussion" below).



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Fig. 3.   Identification of a 22-kDa peflin-interacting protein as ALG-2. Whole Jurkat cell extracts in the presence of 5 mM EGTA (E) or 0.01 mM CaCl2 (C) were immunoprecipitated (IP) with MoAb 32C10A (control), MoAb P1G (peflin), or anti-ALG-2 PoAb (ALG-2). Co-immunoprecipitated proteins (Ppt) and cell lysates (Lysate) were detected by Western blotting (WB) using respective antibodies. Comparable amounts of the relevant samples were adjusted with volumes.

Next, we examined the interaction between the two PEF proteins using HEK293 cells co-transfected with FLAG-tagged peflin and ALG-2 expression vectors. ALG-2 was detected in the immunoprecipitates of FLAG-peflin using anti-FLAG MoAb M2 (Fig. 4A). ALG-2 was also co-immunoprecipitated with N-terminal truncated peflin (FLAG-peflinDelta N), indicating that the PEF domain of peflin is the site of this interaction. This was confirmed by complementary co-immunoprecipitation of FLAG-ALG-2 with untagged peflin (Fig. 4B). On the other hand, untagged peflin was not co-immunoprecipitated with FLAG-peflin (Fig. 4C), suggesting no possibility of peflin/peflin interaction. In contrast, untagged ALG-2 was precipitated with FLAG-tagged ALG-2 regardless of the presence of either EGTA or CaCl2 as reported previously (Fig. 4D and Ref. 20).



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Fig. 4.   Examination of peflin/ALG-2 interaction. HEK293 cells were co-transfected with expression vectors as indicated. A, FLAG-peflin or FLAG-peflinDelta N, and ALG-2. B, FLAG-ALG-2 and peflin. C, FLAG-peflin and peflin. D, FLAG-ALG-2 and ALG-2. After 48 h, cells were lysed in the presence of 5 mM EGTA (E) or 0.01 mM CaCl2 (C) and immunoprecipitated (IP) with anti-FLAG MoAb M2. The immunoprecipitates were analyzed by Western blotting (WB) with anti-peflin MoAb P1G or anti-ALG-2 PoAb. Mouse immunoglobulin light chains (IgG-L) of MoAb M2 in the immunoprecipitates were also detected by subsequent Western blotting using peroxidase-conjugated anti-mouse IgG as a secondary antibody.

Gel Filtration Analysis of Peflin/ALG-2 Heterodimer-- To further examine whether peflin forms a complex with ALG-2, we performed gel chromatography of the soluble fraction of Jurkat cells in the presence of EGTA. Peflin and ALG-2 were co-eluted in the fractions corresponding to 40-50 kDa (Fig. 5B, top and middle, fractions 9-14) greater than the calculated molecular masses (peflin, 30 kDa; ALG-2, 22 kDa). On the other hand, recombinant human ALG-2 was detected in the fractions eluting later than in those by Jurkat ALG-2 (Fig. 5B, bottom, fractions 13-16). Recombinant human peflin was insoluble and could not be applied to the column.



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Fig. 5.   Co-elution of peflin and ALG-2 in gel filtration column chromatography. Preparation of the soluble fraction of Jurkat cells and recombinant ALG-2 (rALG-2), and gel filtration were performed as described under "Experimental Procedures." A, 100,000 × g supernatant was applied to a Superdex-75 column (1.0 × 30 cm), and 0.2-ml fractions were collected. The peak positions of three molecular mass markers are indicated with arrows. BSA, bovine serum albumin (67 kDa); OVA, ovalbumin (43 kDa); CHY, chymotrypsinogen (25 kDa). B, Western blotting of each fraction with anti-peflin MoAb P1G or anti-ALG-2 PoAb. Fraction numbers in panel A are indicated above each lane.

Subcellular Localization of Peflin and ALG-2-- We investigated the localization of peflin and ALG-2 using Jurkat cells. Double-immunofluorescent staining was performed using both anti-peflin MoAb P1G and anti-ALG-2 PoAb and analyzed by confocal laser scanning microscopy. Immunofluorescence was detected in the cytoplasm for peflin and in both the cytoplasm and the nucleus for ALG-2 (Fig. 6).



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Fig. 6.   Subcellular localization of peflin and ALG-2 in indirect immunofluorescent staining. Jurkat cells were double-immunostained for peflin (green) with anti-peflin MoAb P1G or for ALG-2 (red) with anti-ALG-2 PoAb by detecting with secondary fluorescein isothiocyanate-conjugated anti-mouse IgG antibody and secondary rhodamine-conjugated anti-rabbit IgG antibody, respectively, as described under "Experimental Procedures." Immunofluorescences were visualized under a confocal laser-scanning microscope, and a merged image was obtained. Scale bar indicates 10 µm.

As shown in Fig. 7A, peflin was recovered in the cytosolic fraction (S) using a lysis buffer containing 3 mM MgCl2 but containing neither EGTA nor CaCl2 by subcellular fractionation based on the differential centrifugation method. In contrast, ALG-2 was recovered in the crude nuclear fraction (P1), as well as in the cytosolic fraction. The crude nuclear fraction was subjected to centrifugation on a 2 M sucrose cushion. ALG-2 was detected in the purified nuclei (N), agreeing with the result of immunofluorescent staining (Fig. 6). In the presence of 0.01 mM CaCl2, however, almost all peflin and ALG-2 were recovered in the crude nuclear fraction (Fig. 7, B and C, P1), but the purified nuclei contained a smaller amount of peflin and most of the peflin protein was recovered in the membrane/cytoskeletal fraction above a 2 M sucrose cushion (data not shown). In contrast, under the same conditions, roughly equal amounts of ALG-2 were recovered in the membrane/cytoskeletal fraction and in the purified nuclei (data not shown). Inclusion of 0.1% Triton X-100 in a buffer containing 5 mM EGTA or 0.01 mM CaCl2 partially solubilized peflin and ALG-2 (Fig. 7, B and C), but some of the PEF proteins were resistant to the detergent. A higher concentration of Triton X-100 (1%) gave similar results (data not shown). These results suggested that peflin and ALG-2 also changed their subcellular distribution with Ca2+ as reported for other PEF proteins, probably from the cytosol to the membrane and detergent-insoluble (cytoskeletal) fractions.



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Fig. 7.   Subcellular fractionation of peflin and ALG-2. A, Jurkat cells were homogenized in a low salt buffer and fractionated into cytosolic (S), light membrane (P3), heavy membrane (P2), and crude nuclear (P1) fractions by the differential centrifugation method as described under "Experimental Procedures." The crude nuclear fraction was re-homogenized and centrifuged on a 2 M sucrose cushion to obtain the purified nuclear fraction (N). Volumes for SDS-PAGE were adjusted to compare the relative amounts of the PEF proteins in the subcellular fractions. Peflin and ALG-2 were detected by Western blotting using anti-peflin P1G or anti-ALG-2 PoAb. B, effects of Ca2+ on subcellular fractionation of peflin and ALG-2 were examined. Cellular fractionation into cytosol (S), membrane (P2/P3), and crude nuclear (P1) fractions was performed in the presence of 5 mM EGTA or 0.01 mM CaCl2. Effects of a non-ionic detergent, 0.1% Triton X-100, on solubilization of peflin and ALG-2 were examined. C, the immunoblots shown in Fig. 7B were scanned with a flat-bed scanner (EPSON GT-7000 ART), and the densities were quantified using a computerized image analysis system for Macintosh (NIH Image software, version 1.55). Relative amounts of peflin and ALG-2 in each subcellular fraction were expressed in histograms, respectively, where the sum of each fraction (S, P2/P3 and P1) is 1.0.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We showed that peflin exists as a complex with ALG-2 in the absence of Ca2+ but that the complex dissociates in the presence of the divalent cation. Peflin and ALG-2 seem to interact directly because the two proteins were eluted from the gel filtration column at the position of a heterodimer (Fig. 5). Previously, Missotten and colleagues (20) showed that ALG-2 forms a homodimer by co-immunoprecipitation of transiently co-overexpressed FLAG- and Myc-tagged proteins in HEK293 cells. In agreement with their result, we also detected a complex of exogenously expressed FLAG-tagged ALG-2 and untagged ALG-2, but the efficiency of the dimer formation was lower than that of the heterodimer formation with peflin (Fig. 4, B and D). Without co-transfection with untagged ALG-2, no ALG-2 immunoreactive band was detected in the immunoprecipitates of anti-FLAG MoAb M2 (data not shown). In similar experiments, no FLAG-peflin/peflin complex was observed (Fig. 4C). On the other hand, a peflin/ALG-2 complex was clearly observed in the co-transfection assays (Fig. 4, A and B) and even in the endogenously expressing proteins in Jurkat cells (Fig. 3). Thus, the formation of a peflin/ALG-2 heterodimer seems dominant over an ALG-2/ALG-2 homodimer.

Since the 22-kDa protein that was co-immunoprecipitated with peflin in 35S-labeled Jurkat cells was identified as ALG-2 (Fig. 2), it became possible to estimate an approximate molar ratio between peflin and ALG-2 in the complex. Assuming that peflin and ALG-2 incorporate 35S-labeled amino acids with similar efficiencies during de novo synthesis, relative specific radioactivities can be calculated from the numbers of methionine (excluding translation initiation Met) and cysteine residues in the proteins (peflin: 9 Met, 5 Cys; ALG-2: 3 Met, 1 Cys). Thus, the ratio of relative specific radioactivities of [35S]peflin and [35S]ALG-2 is 14:4. This ratio agrees well with that of the observed relative radioactivities of the 30-kDa band (peflin) and the 22-kDa band (ALG-2) in the autoradiogram analyzed by a bioimaging analyzer BAS 2000 system (3.5:1 versus 4.2:1 calculated from photostimulated luminescence units: peflin, 556; ALG-2, 132). This fact indicates the presence of an approximately equal molar ratio (1:0.83) of peflin and ALG-2 in the immunoprecipitates and suggests that the majority of peflin exists as a heterodimer with ALG-2 in the cytosol in the absence of Ca2+.

On the other hand, not all of ALG-2 forms a heterodimer with peflin. Approximately 50% of ALG-2 is present in the 100,000 × g supernatant fraction, and the rest is found in nuclei and membrane/cytoskeletal fractions (Figs. 6 and 7). Because the amounts of peflin in the latter fractions are quite low, non-cytosolic ALG-2 may exist either as a homodimer or complexed with unknown macromolecules. Co-elution of cytosolic ALG-2 with peflin in the gel filtration chromatography suggests that the majority of cytosolic ALG-2 forms a heterodimer with peflin (Fig. 5). The results of the immunoprecipitation experiments using anti-ALG-2 PoAb, however, do not support this notion (Fig. 3). Whereas only a fraction (<10%) of ALG-2 was immunoprecipitable from the lysate, more than half of peflin was co-immunoprecipitable with the antibody. The major cause of this inconsistency may be due to the nature of the anti-ALG-2 PoAb used in this study. The antibody was first raised in rabbits using denatured recombinant mouse ALG-2 and was later affinity-purified using recombinant human ALG-2 as a ligand. The obtained antibody may recognize only a fraction of ALG-2 that retains a specific conformation favoring interaction with peflin and may poorly recognize ALG-2 monomers and homodimers under undenatured conditions. Alternatively, the antibody may disrupt the protein-protein interaction under investigation. It is unlikely that the co-immunoprecipitation of peflin with anti-ALG-2 PoAb was due to a cross-reactivity of the antibody with peflin, because the antibody did not react with peflin overexpressed in HEK293 cells by Western blotting (data not shown). Indeed, anti-FLAG MoAb co-immunoprecipitated untagged peflin together with FLAG-tagged ALG-2 from the lysates of HEK293 cells transfected with the tagged ALG-2-expressing construct (Fig. 4B).

X-ray crystallographic analysis of the PEF domains of the recombinant rat and pig calpain small subunits revealed homodimerization through EF-5 of each molecule (3, 4). Recently, the heterodimers of recombinant m-calpains of the large and small subunits and the homodimer of grancalcin have been crystallized, and PEF domains have been shown to form similar dimer structures through EF-5s (21-23). We assume that peflin forms a heterodimer with ALG-2 by a similar protein-protein interaction mechanism. The N-terminal hydrophobic region of peflin is not essential for heterodimer formation as revealed by FLAG-peflinDelta N (Fig. 4A). In the present study, however, we could not investigate the potential role of the EF-5 domains of peflin and ALG-2 in their interaction. A deletion mutant lacking EF-5 (peflinDelta EF5) could not be expressed in transient transfection experiments using HEK293 cells suggesting the importance of heterodimerization with ALG-2 for stability and/or correct folding of peflin.

The results of cellular fractionation experiments suggested that peflin and ALG-2 translocate from the cytosolic fraction to the membrane and Triton X-100-insoluble (cytoskeletal) fractions in a Ca2+-dependent manner (Fig. 7). In this study, however, we could not detect a change in the immunofluorescence after stimulation of Jurkat cells with Ca2+-ionophore, and we could not obtain direct evidence of the Ca2+-induced translocation of these proteins by immunofluorescent staining. Surprisingly, ALG-2 was also found to localize in nuclei, raising the possibility of a specific function in nuclear Ca2+ signaling. Recently, Krebs and Klemenz (24) showed the nuclear localization of ALG-2 by immunofluorescent staining of breast cancer cells and observed disappearance of the nuclear localization of ALG-2 at the onset of mitosis.

Apoptotic pathways of ALG-2 have been partially clarified. ALG-2 was originally identified by the method called "death trap" in T-cell hybridoma using anti-CD3 antibody (7). An antisense ALG-2 cDNA expression prompted survival after a variety of apoptotic stimuli, but caspase activities were not affected (25). An ALG-2-interacting protein named either AIP1 (ALG-2-interacting protein 1) or Alix (ALG-2-interacting protein X) was cloned concurrently by two independent groups (20, 26). AIP1 is a 105-kDa protein with a proline-rich C-terminal region containing 10 PXXP sequence motifs that potentially bind to SH3 domains. The N-terminal-truncated AIP1 construct exerted dominant-negative effects on the apoptosis of transfected cells induced by starvation of trophic factors or staurosporine (26). The interaction between AIP1 and ALG-2 requires Ca2+. In addition, AIP1 has been reported to interact with SETA (SH3 domain-containing protein expressed in tumorigenic astrocytes) through its C-terminal proline-rich region, which binds to SH3-N (one of the two SH3 domains) of SETA in a Ca2+-independent manner (27). Overexpressed SETA proteins capable of binding to AIP1 sensitized astrocytes to UV light-induced cell death. Thus, in resting cells, SETA/AIP1 and peflin/ALG-2 complexes may exist separately in the cytoplasm. After Ca2+-mobilization, ALG-2 may dissociate from peflin and interact with SETA/AIP1 complex. In our preliminary experiments, however, peflin-overexpressed HEK293 cells did not show morphological changes and differences in apoptotic sensitivity upon stimulation with Ca2+-ionophore or staurosporine compared with control transfectants. Studies are in progress to investigate the potential role of peflin in Ca2+-dependent apoptosis under various conditions.


    ACKNOWLEDGEMENTS

We thank H. Shibata for his valuable suggestions, T. Kawai for his technical assistance, and Dr. K. Matsumoto for protocols of HEK293 cell transfection experiments.


    FOOTNOTES

* This work was partly supported by Grant-in-aid for Scientific Research (Category B), 11460038 from the Ministry of Education, Science, Sports, and Culture of Japan (to M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-52-789-4088; Fax: 81-52-789-5542; E-mail: mmaki@agr.nagoya-u.ac.jp.

Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M008649200


    ABBREVIATIONS

The abbreviations used are: PEF, penta-EF-hand; MoAb, monoclonal antibody; HEK, human embryonic kidney; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PoAb, polyclonal antibody; SETA, SH (Src homology) 3 domain-containing protein expressed in tumorigenic astrocytes.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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