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(The FASEB Journal. 2000;14:1083-1092.)
© 2000 FASEB

Proteolytic cleavage of phospholipase C-{gamma}1 during apoptosis in Molt-4 cells

SUN SIK BAE*, DAVID K. PERRY{dagger}, YONG SEOK OH*, JANG HYUN CHOI*, SEHAMUDDIN H. GALADARI{ddagger}, TARIQ GHAYUR§, SUNG HO RYU*, YUSUF A. HANNUN{dagger} and PANN-GHILL SUH*1

* Department of Signal Transduction, Division of Molecular and Life Science, Pohang University of Science and Technology, Kyungbuk, Pohang 790–784, Republic of Korea;
{dagger} Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA;
{ddagger} Department of Biochemistry, Faculty of Medicine and Health Science, UAE University, Al Ain, UAE; and
§ BASF Bioresearch Corporation, Worcester, Massachusetts 01605, USA

1Correspondence: Department of Signal Transduction, Division of Molecular and Life Science, Pohang University of Science and Technology, San 31 Hyoja-Dong, Nam-Gu, Kyungbuk, Pohang 790–784, Republic of Korea. E-mail: pgs{at}pop.postech.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospholipase C-{gamma}1 (PLC-{gamma}1), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leukemic Molt-4 cells with PLC inhibitors such as U-73122 or ET-18-OCH3 potentiated etoposide-induced apoptosis in these cells. PLC-{gamma}1 was fragmented when Molt-4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor {alpha}. Cleavage of PLC-{gamma}1 was blocked by overexpression of Bcl-2 and by specific inhibitors of caspases such as Z-DEVD-CH2F and YVAD-cmk. Purified caspase-3 and caspase-7, group II caspases, cleaved PLC-{gamma}1 in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC-{gamma}1 is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala-Glu-Pro-Asp770 was identified to be a cleavage site within PLC-{gamma}1. Epidermal growth factor receptor (EGFR) -induced tyrosine phosphorylation of PLC-{gamma}1 resulted in resistance to cleavage by caspase-3 in vitro. Furthermore, cleaved PLC-{gamma}1 could not be tyrosine-phosphorylated by EGFR in vitro. In addition, tyrosine-phosphorylated PLC-{gamma}1 was not significantly cleaved during etoposide-induced apoptosis in Molt-4 cells. This suggests that the growth factor-induced tyrosine phosphorylation may suppress apoptosis-induced fragmentation of PLC-{gamma}1. We provide evidence for the biochemical relationship between PLC-{gamma}1-mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC-{gamma}1-mediated signaling pathway can facilitate an apoptotic progression.—Bae, S. S., Perry, D. K., Oh, Y. S., Choi, J. H., Galadari, S. H., Ghayur, T., Ryu, S. H., Hannun, Y. A., Suh, P.-G. Proteolytic cleavage of phospholipase C-{gamma}1 during apoptosis in Molt-4 cells.


Key Words: PLC-{gamma}1 • proteolysis • tyrosine phosphorylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
APOPTOSIS, OR PROGRAMMED cell death, is critical to many biological processes, including development of the immune system, elimination of virus-infected cells, and embryogenesis (1) . An important aspect in the elucidation of the biochemical mechanism of apoptosis is the role of proteases. Ced-3 has been defined as a molecular component of cell death by genetic analysis of developmental cell death in Caenorhabditis elegans (2) . Ced-3 encodes a protein that is homologous to the mammalian gene interleukin-1ß-converting enzyme (ICE), which is termed caspase-1 (3) . So far 10 caspase-1 homologs have been isolated (4) , including Nedd2/Ich-1 (caspase-2), CPP32/Apopain/Yama (caspase-3), TX/ICErelII/Ich-2 (caspase-4), TY/ICErelIII (caspase-5), Mch2 (caspase-6), Mch3/ICE-LAP3/CMH-1 (caspase-7), FLICE/MACH/Mch5 (caspase-8), Mch6/ICE-LAP6 (caspase-9), and Mch4 (caspase-10). These proteases share similar structural features: they contain an active site QACRG pentapeptide and are unique in their requirement for an aspartate residue at the P1 position in the cleavage site of substrate proteins (5) . In common with other protease zymogens, generation of an active form requires limited proteolysis. This results from cleavage in an interdomain linker segment to give a heterodimeric enzyme, with both chains containing essential components of the catalytic machinery (6) . Cleavage of proteins by caspases is not only specific, but also highly efficient. The preferred tetrapeptide recognition motif differs significantly among caspases and explains the diversity of their biological functions. Not all proteins containing the optimal tetrapeptide sequence are cleaved, implying that tertiary structural elements may influence substrate recognition. The strict specificity of caspases is consistent with the observation that apoptosis is accompanied by a selective set of protein digestion in a coordinate manner.

On binding of Fas ligand or tumor necrosis factor {alpha} (TNF-{alpha}) to their cognate receptors, caspase-8 is recruited to the death-induced signaling complex through interaction with FADD/MORT-1 (7) . Caspase-8 becomes activated through autocatalysis within this multiprotein complex (8) . Bid is cleaved by caspase-8 and the carboxyl-terminal part of cleaved Bid translocates to the mitochondria, where it triggers cytochrome c release into cytoplasm (9) . Recently, it has been shown that pro-apoptotic Bcl-2 family members such as Bax and Bak accelerate the opening of voltage-dependent anion channel (VDAC), resulting in the release of cytochrome c (10) . It has been revealed that dATP together with cytochrome c associates with Apaf-1, which is a mammalian homologue of CED-4 (11) . Activated Apaf-1 subsequently associates with caspase-9 and induces activation of caspase-3 (12) . Activation of caspase-3 leads to inactivated proteins, which protect living cells from apoptosis. A clear example is the cleavage of ICAD/DFF45 (13) , an inhibitor of the nuclease responsible for DNA fragmentation, CAD (caspase-activated deoxyribonuclease). During apoptosis, ICAD is inactivated by caspases, leaving CAD free to function as a nuclease. Other target proteins of caspases are the Bcl-2 family proteins (14) . It appears that cleavage not only inactivates these proteins, but also produces a fragment that promotes apoptosis. Caspases also influence the morphological changes of apoptotic cells by cleaving several proteins involved in cytoskeletal regulation, including gelsolin (15) , focal adhesion kinase (16) , and p21-activated kinase 2 (17) .

It has been reported that growth promoting agonists such as epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor-1 (IGF-1), and cytokines can suppress apoptosis (18) . These growth factor receptors activate several key signaling molecules including phosphatidylinositol 3-kinase (PI3K), small molecular weight G-protein such as Ras, and phospholipase C-{gamma} (PLC-{gamma}). Phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), which is generated by PI3K, activates PKB/Akt and PLC-{gamma}1 (19 , 20) . PLC-{gamma}1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers: diacylglycerol, an activator of protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which mobilizes Ca2+ from an intracellular Ca2+ store (21) . Besides the role of PLC-{gamma}1 in the generation of second messengers, the src homology domain (SH2 and SH3) of PLC-{gamma}1 is implicated in mitogenic signaling (22) . Like other growth signaling molecules such as PI3K and Ras, it has been revealed that PLC-{gamma}1 has transforming activity (23) and seems to play central roles in cellular proliferation.

In this study, we show for the first time that PLC-{gamma}1 is a substrate of group II caspases. Cleaved PLC-{gamma}1 cannot be phosphorylated by epidermal growth factor receptor (EGFR). The PLC-{gamma}1 phosphorylated at tyrosine residues by growth factor receptor kinase is resistant to proteolytic cleavage. These results suggest that the defect of PLC-{gamma}1-mediated signaling may result in an increased apoptotic progression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General reagents
Tissue culture supplies were purchased from Corning (Corning, N.Y.) and sera were from Hyclone (Logan, Utah). Etoposide and anti-FLAG M5 monoclonal antibody were purchased from Sigma (St. Louis, Mo.). Z-DEVD-CH2F was purchased from Enzyme Systems Products (Dublin, Calif.). YVAD-cmk, TNF-{alpha}, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), and hygromycin B were obtained from Calbiochem Corp. (San Diego, Calif.). An enhanced chemiluminescence (ECL) detection system was purchased from Amersham Corp. (Aylesbury, U.K.). Various ceramide analogs were prepared by Alicja Bielawska as described (24) . Monoclonal anti-PLC-{gamma}1 antibody was generated as described previously (25) . Rabbit polyclonal antibody raised to Escherichia coli expressed caspase-3 was prepared by immunization of New Zealand white rabbits as described previously (26) . Caspases are prepared as described previously (27) and caspase activity was measured using specific peptide substrate. Anti-phosphotyrosine antibody (4G10) was purchased from Upstate Biotech. Inc. (Lake Placid, N.Y.). Peroxidase-conjugated anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG were purchased from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, Md.).

Cell culture
Molt-4 cells were purchased from ATCC (Rockville, Md.), and cultured in RPMI 1640 medium with 10% heat-inactivated bovine calf serum, and maintained at 5% CO2/95% air at 37°C. Molt-4 cells were transfected with full-length murine bcl-2 gene as described previously (28) . For experiments, cells were plated at a density of 2.5 x 106 cells/ml in RPMI 1640 with 2% bovine calf serum and treated with etoposide (20 µM), each ceramide analog (10 µM), TNF-{alpha} (100 ng/ml), or vehicle alone as control. For the inhibitor studies, Molt-4 cells were pretreated with ET-18-OCH3 (10 µM), U-73122 (10 µM), YVAD-cmk, or Z-DEVD-CH2F for 30 min before etoposide treatment.

Expression and purification of PLC-{gamma}1
Spodoptera frugiperda (Sf9) cells were cultured at 27°C in TC-100 medium (Life Technologies, Inc.-BRL) supplemented with 10% fetal calf serum. cDNA of PLC-{gamma}1 was subcloned into pVL1393 (Invitrogen). Sf9 cells were cotransfected with recombinant pVL1393/PLC-{gamma}1 and BaculoGold (PharMingen, San Diego, Calif.) using FuGene transfection reagent (Boehringer Mannheim, Mannheim, Germany). For large-scale expression, recombinant baculovirus was amplified and infected into Sf9 cells as described previously (29) . Cells were harvested and washed with phosphate-buffered saline (PBS) and then lysed with lysis buffer (20 mM HEPES-OH pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 0.1 mM dithiothreitol). PLC-{gamma}1 was purified using sequential DEAE-5PW, phenyl-5PW, and heparine-5PW column chromatography (TosoHaas, Tokyo).

Phosphorylation of PLC-{gamma}1 in vitro
Purified PLC-{gamma}1 was phosphorylated by epidermal growth factor receptor (EGFR) purified by wheat germ lectin agarose from A431 cells. Three hours after incubation in a kinase buffer (20 mM HEPES-OH pH 7.4, 25 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3VO4, and 0.3 µM epidermal growth factor), phosphorylated and unphosphorylated PLC-{gamma}1 were separated by anti-phosphotyrosine antibody-conjugated protein A Sepharose. Phosphorylated PLC-{gamma}1 and EGFR were eluted by 50 mM phenyl phosphate. Phosphorylated EGFR was discarded by precipitation using wheat germ lectin agarose. Phosphorylated PLC-{gamma}1 fraction was applied to gel filtration column to exchange buffer into cleavage assay buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 3 mM NaCl, 0.2 µM CaCl2, and 1 mM DTT).

In vitro PLC-{gamma}1 cleavage and in vitro PLC activity
Cleavage of PLC-{gamma}1 was performed by active caspases as described previously (30) . Briefly, reactions were initiated by the addition of various caspases to 1 µg of purified PLC-{gamma}1 in a total volume of 15 µl containing 20 mM HEPES, pH 7.4, 100 mM KCl, 3 mM NaCl, 0.2 µM CaCl2, and 1 mM DTT. After the indicated times, the reactions were stopped by adding SDS sample buffer. After treating PLC-{gamma}1 with caspase-3 at the times indicated, PLC activity was assayed with [3H]phosphatidylinositol as substrate. The lipids in chloroform were dried under a stream of nitrogen gas, suspended in 0.4% sodium deoxycholate, and subjected to sonication. Enzyme activity was quantitated in the 200 µl assay mixture containing 150 µM PI (20,000 cpm [3H]PI), 1 mM EGTA, 10 mM CaCl2, 0.1% sodium deoxycholate, and 50 mM HEPES, pH 7.0. The reaction mixture was incubated at 37°C and the reaction was stopped by adding 1 ml of chloroform:methanol:HCl (100:100:0.6, v/v/v), followed by 0.3 ml of 1 N HCl containing 5 mM EGTA. After centrifugation, 0.5 ml of the upper aqueous phase was assayed for radioactivity by liquid scintillation counter.

Plasmid construction and mutagenesis
Rat PLC-{gamma}1 cDNA was amino-terminally tagged with FLAG sequence and subcloned into pFLAG-CMV (pFLAG-CMV/{gamma}1). The D485N mutation was made by a splice-overlap extension method (31) . Briefly, we first obtained polymerase chain reaction (PCR) products using forward primer 5' ATG GCG GGC GTC GGG A 3'/reverse primer 5' GAG TTA CTG ATG TTA TTC TCA GAG (D485N) and forward primer 5' CTC TGA GAA TAA CAT CAG TAA CTC 3' (D485N)/reverse primer 5' CTA GAG GCG GTT GTC TAA A 3', respectively. Next, we amplified D485N mutant DNA using forward primer 5'ATG GCG GGC GTC GGG A 3' and reverse primer 5' CTA GAG GCG GTT GTC TAA A 3'. From the large PCR product, MluI/BstEII fragment containing the mutation was removed and cloned back into the wild-type sequence. The region corresponding to the MluI/BstEII fragment was fully sequenced to ensure that no other mutation was introduced inadvertently. A similar strategy was used to generate D732A and D770A mutations using the internal oligonucleotides as follows: forward primer 5' TCT GAG TTT GCC AGC CTG GTC 3'/reverse primer 5' GAC CAG GCT GGC AAA CTC AGA G-3' for D732A, and forward primer 5' GCT GAA CCC GCT TAT GGG GCA 3'/reverse primer 5' TGC CCC ATA AGC GGG TTC AGC-3' for D770A.

Immunoprecipitation and immunoblotting
Cells were treated with etoposide (20 µM) for the times indicated, washed twice with PBS, and lysed in the lysis buffer (20 mM HEPES, pH 7.2, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 0.1 mM DTT, 1 µg/ml Leupeptin, and 1% Triton X-100). After sonication, the cell homogenates were centrifuged at 10,000 g for 10 min. Fifty micrograms of cell lysate were electrophoresed on 8% polyacrylamide gels for immunoblotting of PLC-{gamma}1 or 12% polyacrylamide gels for immunoblotting of caspase-3. For immunoprecipitation, 400 µg of cell lysates were immuoprecipitated with either anti-phosphotyrosine or anti-PLC-{gamma}1 antibody-conjugated protein A agarose. The immunoprecipitates were subjected to SDS-PAGE and immunoprobed with either anti-PLC-{gamma}1 or anti-phosphotyrosine antibody. The blots were developed with ECL reagents and the signal was detected by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of PLC activity potentiates etoposide-induced apoptosis in Molt-4 cells
Several reports have demonstrated that PKC serves as an activator of Bcl-2 and eventually plays an anti-apoptotic role (32) . PLC modulates PKC activity by controlling the level of intracellular diacylglycerol and Ca2+ (21) . Pretreatment of T leukemic Molt-4 cells with PLC inhibitors such as U-73122 or ET-18-OCH3 potentiated the etoposide-induced cell death in a time-dependent manner (Fig. 1 ). However, U-73122 or ET-18-OCH3 alone did not cause significant cell death in Molt-4 cells. Also, PLC inhibitors potentiated etoposide-induced DNA fragmentation (S. S. Bae et al., unpublished observations).



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  		Figure 1. Effect of PLC inhibitors on the etoposide-induced apoptosis. Molt-4 cells were pretreated with either U-73122 or ET-18-OCH3 for 20 min and then treated with etoposide for indicated times. Dead cells were counted using a trypan blue exclusion method. Data are the mean ± SD of three independent experiments.

PLC-{gamma}1 is proteolytically cleaved during apoptosis
Various extracellular stimuli such as Fas, TNF-{alpha}, and chemotherapeutic agents induce apoptosis in hemopoietic cell lines. Treatment of Molt-4 cells with etoposide, TNF-{alpha}, and ceramides induced proteolytic cleavage of PLC-{gamma}1 and generated a 60 kDa cleavage product detectable by monoclonal anti-PLC-{gamma}1 antibody, as shown in Fig. 2 . Among the PLC isozymes tested, PLC-{gamma}1 and PLC-{delta}3 were predominantly expressed in Molt-4 cells. However, PLC-{delta}3 was not cleaved by etoposide treatment (S. S. Bae et al., unpublished observations).



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  		Figure 2. Various apoptotic stimuli-induced cleavage of PLC-{gamma}1. A) Molt-4 cells were treated with etoposide (20 µM) and each ceramide analog (10 µM) for 9 h. Cells were washed twice with PBS and proteolytic cleavage of PLC-{gamma}1 was probed by anti-PLC-{gamma}1 antibody. B) Molt-4 cells were treated with TNF-{alpha} (100 ng/ml) in the presence or absence of cycloheximide (CHX) (10 µg/ml). The cells were washed twice with PBS and the cell lysates were immunoprobed with anti-PLC-{gamma}1 antibody (upper panel) or anti-caspase-3 antibody (lower panel). Etoposide (20 µM) treated cell lysates were included as a control.

Induction of apoptosis and activation of caspases by TNF-{alpha} requires protein synthesis inhibitor such as cycloheximide (33) . As shown in Fig. 2B , both TNF-{alpha}-induced activation of caspase-3 and proteolytic cleavage of PLC-{gamma}1 required pretreatment with cycloheximide.

Cleavage of PLC-{gamma}1 is inhibited by overexpression of bcl-2 and inhibitors of caspases
Bcl-2 plays anti-apoptotic function by controlling the activation of caspases (34) . To determine whether cleavage of PLC-{gamma}1 is induced by Bcl-2-dependent caspases, we examined the proteolytic cleavage of PLC-{gamma}1 in Bcl-2-overexpressing cells. Overexpression of the bcl-2 gene completely blocked the etoposide-induced cleavage of PLC-{gamma}1 as shown in Fig. 3A . We pretreated Molt-4 cells with the caspase-1 inhibitor YVAD-cmk and group II caspase inhibitor Z-DEVD-CH2F. Both YVAD-cmk and Z-DEVD-CH2F completely inhibited etoposide-induced proteolytic cleavage of PLC-{gamma}1 at a concentration of 20 µM (Fig. 3B ). However, YVAD-cmk required a higher concentration than DEVD-CH2F to block the cleavage of PLC-{gamma}1 and activation of pro-caspase-3 (Fig. 3C ). Since the intact form of PLC-{gamma}1 was recovered in a caspase inhibitor-dependent manner, it is plausible that fragmentation of PLC-{gamma}1 is caspase dependent. Also, pretreatment of PLC inhibitors strongly potentiated the cleavage of PLC-{gamma}1 as well as activation of caspase-3 (S. S. Bae et al., unpublished observations). These results indicate that PLC may inhibit apoptotic protease activity and that the cleavage of PLC-{gamma}1 is more sensitive to Z-DEVD-CH2F, group II caspase inhibitor than to YVAD-cmk, caspase-1 inhibitor.



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  		Figure 3. Effect of bcl-2 overexpression and caspase-specific inhibitors on the cleavage of PLC-{gamma}1. Molt-4 cells, which were transfected with pMEP4 (vector) and pMEP4/bcl-2, were treated with either etoposide (20 µM) or vehicle for 9 h, and the proteolytic cleavage of PLC-{gamma}1 was detected by anti-PLC-{gamma}1 monoclonal antibody (A). Molt-4 cells were pretreated with either YVAD-cmk or Z-DEVD-CH2F at the indicated concentrations for 30 min, then cells were treated with etoposide (20 µM) for 9 h. 75 µg of cell lysate were immunoblotted with either anti-PLC-{gamma}1 monoclonal antibody (B) or anti-caspase-3 polyclonal antibody (C).

PLC-{gamma}1 is cleaved by group II caspase in vitro
Based on the report by Thornberry et al. (35) , caspases are categorized into three groups (group I: caspase-1, -4, and -5; group II: caspase-3, -7, and -2; group III: caspase-6, -8, and -9). To investigate which group of caspases is involved in the cleavage of PLC-{gamma}1, we incubated purified PLC-{gamma}1 with the active form of caspases. Cleavage of PLC-{gamma}1 was only susceptible to caspase-3 and caspase-7 (Fig. 4A ). Cleavage of PLC-{gamma}1 by caspase-3 and caspase-7 was significantly blocked by treatment of DEVD-CH2F. However, inhibition of PLC-{gamma}1 cleavage by caspase-3 and caspase-7 by YVAD-cmk was nearly negligible (Fig. 4B ). PLC-{gamma}1 was cleaved by caspase-3 in vitro and gave rise to a cleavage product of the same size as that observed in vivo (Fig. 4C ). The cleavage was started at 30 min and nearly all PLC-{gamma}1 was cleaved after 4 h incubation. There was no significant difference in the kinetics of PLC-{gamma}1 cleavage between caspase-3 and caspase-7 (S. S. Bae et al., unpublished observations). The cleavage of PLC-{gamma}1 by caspase-3 did not affect its enzymatic activity (Fig. 4D ). These results strongly indicate that specific caspases cleave PLC-{gamma}1 and that PLC-{gamma}1 could be a direct target of group II caspases in vivo during apoptosis.



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  		Figure 4. Cleavage of PLC-{gamma}1 by caspases in vitro. A) 1 µg of purified PLC-{gamma}1 was incubated with 300 ng of each purified caspase-4, -1, -7, -3, -6, -8, and -2 for 2 h. Reactions were stopped by adding Laemmli buffer, and proteolytic cleavage of PLC-{gamma}1 was detected by anti-PLC-{gamma}1 monoclonal antibody. B) Purified PLC-{gamma}1 was incubated with either caspase-3 or caspase-7 in the presence or absence of caspase inhibitors. Proteolytic cleavage of PLC-{gamma}1 was detected by immunoprobing with anti-PLC-{gamma}1 antibody. C) 1 µg of purified PLC-{gamma}1 was incubated with 300 ng of purified active caspase-3 for the indicated time at 37°C. The reaction conditions were described in Materials and Methods. The reaction mixtures were either stopped by adding Laemmli buffer for the immunoblotting of PLC-{gamma}1 or PLC activity was measured as described in Materials and Methods (D). Data are the mean ± SD of triplicated experiments.

Identification of cleavage site
Although there are no optimal target tetrapeptide sequences (DEXD) for group II caspases in the amino acid sequence of PLC-{gamma}1, we found three possible cleavage sites (XEXD): IELD (485), SEFD (732), and AEPD (770). To identify the cleavage site, both wild-type and mutant PLC-{gamma}1 were tagged at NH2 terminus with FLAG epitope. The point mutants of these sites as well as wild-type PLC-{gamma}1 were transiently expressed in COS-7 cells, and cell extracts were incubated with the active caspase-3. Cleavage of the wild-type and mutant PLC-{gamma}1 was detected by probing with anti-FLAG monoclonal antibody. D485N or D732A mutants were cleaved by caspase-3 and generated a 90 kDa cleavage product (Fig. 5A ). However, mutation at D770A significantly blocked the cleavage by caspase-3. Also, D770A mutant was resistant to cleavage by caspase during TNF-{alpha}/CHX-induced apoptosis in HeLa cells (Fig. 5B ). Neither a significant decrease in the amount of intact protein nor an increase in the cleavage product was observed in D770A mutant. If caspases cleave Asp (770), it may generate NH2-terminal (amino acid 1–770, 90 kDa) and carboxyl-terminal (amino acid 771-1290, 60 kDa) fragments (Fig. 5C ). The size of the fragments detected by anti-FLAG antibody, which recognizes the amino-terminal end of the expressed PLC-{gamma}1, and by anti-PLC-{gamma}1 antibody (B16–5), which recognizes SH3 domain of PLC-{gamma}1 (36) , correlated with the predicted fragment sizes.



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  		Figure 5. Identification of cleavage site. A) D485N, D732A, and D770A mutants were constructed as described in Materials and Methods. Each mutant was transiently transfected into COS-7 cells by electroporation. Cells were harvested 2 days after transfection and the cytosolic fraction of each transfectant was incubated with caspase-3 for 2 h in vitro. Proteolytic cleavage product of PLC-{gamma}1 and its mutants were detected by anti-FLAG monoclonal antibody. B) Each mutant was transiently transfected into HeLa cells and cells were treated with either vehicle alone or TNF-{alpha}/CHX. Proteolytic cleavage of each mutant PLC-{gamma}1 was detected by immunoprobing of each cell lysates with anti-FLAG monoclonal antibody. C) Linear structure of FLAG-tagged PLC-{gamma}1 and the location of a possible cleavage site. Recognition sites by anti-FLAG and anti-PLC-{gamma}1 monoclonal antibodies were indicated by an arrow. The possible fragment sizes detected by either anti-FLAG or anti-PLC-{gamma}1 monoclonal antibody were indicated by a solid or dotted arrow, respectively.

Cleaved PLC-{gamma}1 cannot be phosphorylated by EGFR in vitro
PLC-{gamma}1 is tyrosine-phosphorylated and activated by various receptor tyrosine kinases and by nonreceptor tyrosine kinase (37) . To verify whether cleaved PLC-{gamma}1 is tyrosine-phosphorylated by EGF receptor kinase or not, cleaved PLC-{gamma}1 by caspase-3 was incubated with purified EGF receptor in the presence or absence of ATP. Intact PLC-{gamma}1 was tyrosine-phosphorylated, whereas cleaved PLC-{gamma}1 could not be tyrosine-phosphorylated by EGF receptor as shown in Fig. 6B . To confirm whether the diminish in the tyrosine phosphorylation of cleaved PLC-{gamma}1 was due to the defect in association with EGFR, we incubated both intact and cleaved PLC-{gamma}1 with either phosphorylated EGFR (pEGFR) or unphosphorylated EGFR (uEGFR). Association of PLC-{gamma}1 with EGFR was detected by immunoprobing immunoprecipitates with anti-PLC-{gamma}1 antibody after immunoprecipitation with anti-EGFR antibody. As shown in Fig. 6D , association of cleaved PLC-{gamma}1 as well as intact PLC-{gamma}1 with EGFR was significantly enhanced by autophosphorylation of EGFR.



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  		Figure 6. Effect of PLC-{gamma}1 cleavage on the EGFR-mediated tyrosine phosphorylation. Purified PLC-{gamma}1 was incubated with either buffer alone as control or caspase-3 for 1 h. Caspase-3 was inactivated by the irreversible inhibitor DEVD-CH2F (20 µM). Each sample was incubated with EGFR in the presence or absence of ATP (50 µM) for 2 h. Reactions were stopped by adding sample buffer and proteolytic cleavage and tyrosine-phosphorylation of PLC-{gamma}1 was detected by immunoblotting with anti-PLC-{gamma}1 antibody (A) and anti-phosphotyrosine antibody (B), respectively. Intact PLC-{gamma}1 (Intact-PLC-{gamma}1) and cleaved PLC-{gamma}1 (cleaved-PLC-{gamma}1) by caspase-3 were prepared by treatment of purified PLC-{gamma}1 with either vehicle alone or active caspase-3 for 1 h, respectively. Proteolytic cleavage of PLC-{gamma}1 was confirmed by immunoblotting with anti-PLC-{gamma}1 antibody (C). Reaction mixtures were incubated with either phosphorylated EGFR (pEGFR) or unphosphorylated EGFR (uEGFR) for 1 h. After immunoprecipitation of reaction mixtures with anti-EGFR monoclonal antibody, association of PLC-{gamma}1 with EGFR, amount of EGFR, and tyrosine phosphorylation of EGFR were detected by immunoprobing with anti-PLC-{gamma}1 antibody, anti-EGFR antibody, and anti-phosphotyrosine antibody, respectively (D).

Tyrosine phosphorylation by epidermal growth factor receptor inhibits the cleavage of PLC-{gamma}1 by caspase-3 in vitro
It has been reported that EGFR and platelet-derived growth factor receptor stimulate tyrosine phosphorylation of PLC-{gamma}1 at Tyr771, Tyr783, and Tyr1254 (38) . Since Tyr771 is adjacent to the cleavage site Asp770 of caspase-3, it is possible that phosphorylation at Tyr771 may affect the cleavage of PLC-{gamma}1. To determine whether tyrosine phosphorylation at Tyr771 affects the cleaving action of caspase-3, PLC-{gamma}1 was tyrosine-phosphorylated by EGFR in vitro and the phosphorylated PLC-{gamma}1 (pPLC-{gamma}1) was isolated by sequential use of anti-phosphotyrosine antibody-conjugated protein A Sepharose and wheat germ lectin agarose (Fig. 7A ). Unphosphorylated PLC-{gamma}1 (uPLC-{gamma}1) was rapidly cleaved by caspase-3 in a time-dependent manner (Fig. 7B ). In contrast, pPLC-{gamma}1 was not cleaved by caspase-3.



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  		Figure 7. Effect of tyrosine phosphorylation on the cleavage of PLC-{gamma}1 by caspase-3. A) Phosphorylated (pPLC-{gamma}1) and unphosphorylated PLC-{gamma}1 (uPLC-{gamma}1) were separated as described in Materials and Methods. Both pPLC-{gamma}1 and uPLC-{gamma}1 were immunoblotted with either anti-PLC-{gamma}1 antibody (upper) or anti-phosphotyrosine antibody (lower). B) Both pPLC-{gamma}1 and uPLC-{gamma}1 were incubated with caspase-3 (300 ng) and reaction mixtures were stopped by adding Laemmli buffer. Proteolytic cleavage of PLC-{gamma}1 was detected by anti-PLC-{gamma}1 antibody and anti-phosphotyrosine antibody.

Tyrosine-phosphorylated PLC-{gamma}1 is more resistant to etoposide-induced cleavage in vivo
Our data showed that phosphorylated PLC-{gamma}1 is not susceptible to caspase-3 in vitro. To confirm the susceptibility of tyrosine-phosphorylated PLC-{gamma}1 in vivo, we treated Molt-4 cells with etoposide (20 µM); the cell lysates were immunoprecipitated with either anti-phosphotyrosine antibody or anti-PLC-{gamma}1 antibody and immunoprobed with anti-PLC-{gamma}1 antibody and anti-phosphotyrosine antibody, respectively. Treatment with etoposide led to proteolytic cleavage of PLC-{gamma}1 and activation of pro-caspase-3 in a time-dependent manner (Fig. 8A ). However, the amount of tyrosine-phosphorylated PLC-{gamma}1 was not significantly changed (Fig. 8B ). Furthermore, pretreatment of the cells with tyrosine phosphatase inhibitor such as Na3VO4 significantly blocked etoposide-induced proteolytic cleavage of PLC-{gamma}1 as well as apoptosis (S. S. Bae et al., unpublished observations).



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  		Figure 8. Effect of etoposide on the cleavage of tyrosine-phosphorylated PLC-{gamma}1 in vitro. A) Molt-4 cells were treated with etoposide (20 µM) for the indicated times and cell lysates were immunoblotted with either anti-PLC-{gamma}1 antibody or anti-caspase-3 antibody. B) Molt-4 cells were treated with etoposide (20 µM) and cell lysates were immunoprecipitated with either anti-PLC-{gamma}1 antibody (upper panel) or anti-phosphotyrosine antibody (lower panel); the pellets were immunoprobed with anti-phosphotyrosine antibody (upper panel) or anti-PLC-{gamma}1 antibody (lower panel).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cross talk between signal transduction pathways is fundamental to the final response of a cell to extracellular stimuli. In this context, different signaling pathways can contribute to enhancement or inhibition of apoptosis and cell survival (18) . Several growth signaling molecules such as PKC and PI3K have been identified as anti-apoptotic proteins (19 , 32) . PLC-{gamma}1 plays central roles in growth factor receptor-mediated signal transduction and activates PKC through second messengers such as diacylglycerol and Ca2+ (21) . In this study, we were interested in determining the relationship between PLC-{gamma}1-mediated signaling pathway and apoptosis. Inhibition of PLC activity resulted in the potentiation of etoposide-induced apoptosis in Molt-4 cells. Since etoposide typically induces DNA fragmentation in leukemic cells (30 , 39) and our results showed that PLC inhibitor alone did not induce cell death, PLC inhibitor may stimulate etoposide-induced apoptotic pathway. Also these results implicate the presumed negative roles of PLC in apoptosis.

A common mechanism in apoptosis is the activation of caspases. Activation of caspases accompanies the proteolytic cleavage of cellular proteins that are involved in the cell cycle, morphology, DNA repair, and signal transduction (5) . In this study, several lines of evidences suggest that PLC-{gamma}1 is proteolytically cleaved by group II caspases during apoptosis. First, proteolytic cleavage of PLC-{gamma}1 was blocked by overexpression of Bcl-2. It has been reported that Bcl-2 prevents apoptosis at a step during the activation of caspase family proteases such as caspase-9 and caspase-3 (34) . Second, both YVAD-cmk, which preferentially inhibits caspase-1, and Z-DEVD-CH2F, a specific inhibitor of group II caspases, inhibited proteolysis of PLC-{gamma}1 at high concentrations (>5 µM), and hence led to the recovery of intact PLC-{gamma}1. However, as can be seen from the amount of intact PLC-{gamma}1, the protection of proteolytic cleavage of PLC-{gamma}1 was more effective by Z-DEVD-CH2F than YVAD-cmk at low concentrations (<0.2 µM). It is interesting that the etoposide-induced proteolytic activation of caspase-3 did not correlate with the recovery of intact PLC-{gamma}1. The most probable situation is that not only do YVAD-cmk and Z-DEVD-CH2F inhibit activation step of caspase-3, but they also directly inhibit its activity. Finally, among various caspases, caspase-3 and caspase-7 were the most effective in cleaving PLC-{gamma}1 in vitro and generated fragments whose size were the same as those observed in apoptotic cells in vivo. Other caspases were not effective. These results strongly support that PLC-{gamma}1 is specifically cleaved by group II caspases, especially by caspase-3 and caspase-7 in vivo.

It has been reported that the optimal target tetrapeptide cleavage sequence of group II caspases is DEXD (35) . There is no DEXD motif in the PLC-{gamma}1 sequence. However, we identified three possible cleavage sequences: IELD485, SEFD732, and AEPD770. Even though the P4 position of the cleavage site is Ala instead of Asp, AEPD770 was determined to be the major cleavage site recognized by caspase-3 in vitro. Since the size of the cleavage product is the same in vitro and in vivo, AEPD sequence can also be a cleavage sequence of group II caspases in vivo.

PLC-{gamma}1 is tyrosine-phosphorylated by several growth factors such as EGF, PDGF, and NGF (38) . The major phosphorylation sites of PLC-{gamma}1 are Tyr771, Tyr783, and Tyr1254. Since Tyr771 is adjacent to cleavage site Asp770, it is possible that tyrosine phosphorylation at Tyr771 may affect the cleavage of PLC-{gamma}1 by caspase-3. Our results demonstrate that tyrosine phosphorylation of PLC-{gamma}1 by EGFR significantly reduces the cleavage by caspase-3 in vitro. This type of protection from caspase-3 by phosphorylating of the residue adjacent to the recognition site of caspase-3 has also been reported for I{kappa}B-{alpha}. Phosphorylation at the serine residue of I{kappa}B-{alpha} inhibits its cleavage (40) . Several data implicate that phosphorylated PLC-{gamma}1 is resistant to cleavage by caspases. First, phosphorylated PLC-{gamma}1 is resistant to cleavage by caspase-3 in vitro. Second, though tyrosine phosphorylation of intact PLC-{gamma}1 was clearly detectable in immunoprecipitates by the anti-PLC-{gamma}1 antibody that recognizes both intact and cleaved PLC-{gamma}1, we could not detect tyrosine phosphorylation in the cleavage product of PLC-{gamma}1. Also, the level of tyrosine phosphorylated PLC-{gamma}1 was not significantly changed during etoposide-induced apoptosis. Third, immunoprecipitation of etoposide-treated Molt-4 cell lysates by anti-phosphotyrosine antibody and subsequent immunoblotting with anti-PLC-{gamma}1 antibody (it recognizes both intact PLC-{gamma}1 and cleavage product of PLC-{gamma}1) did not show detectable cleavage product. Although the overall amount of PLC-{gamma}1 was significantly reduced due to the cleavage, the amount of tyrosine-phosphorylated PLC-{gamma}1 is not significantly reduced during apoptosis in Molt-4 cells. The resistance of the tyrosine-phosphorylated PLC-{gamma}1 to caspases in vivo may be ascribed to its tyrosine phosphorylation at Tyr771 as in vitro. These results indicate that growth factor-induced tyrosine phosphorylation of PLC-{gamma}1 results in the resistance to cleavage by apoptotic proteases. However, we cannot rule out the possibility that resistance to cleavage of phosphorylated PLC-{gamma}1 by caspase may result from the different subcellular localization in vivo. It has been reported that PLC-{gamma}1 translocates from cytosol to membrane by occupation of growth factors on cognate receptor (41) . Since caspase-3 is mainly localized in cytosol (42) , it is possible that caspase-3 cannot cleave membrane-localized PLC-{gamma}1 (it may be a tyrosine-phosphorylated form) in vivo.

Since our results show that only unphosphorylated PLC-{gamma}1 seems to be cleaved by caspases, we checked the effect of cleavage of PLC-{gamma}1 on the tyrosine phosphorylation by growth factor receptor. Though cleaved PLC-{gamma}1 can associate with phosphorylated EGFR, it cannot be tyrosine-phosphorylated by EGFR in vitro. This result indicates that proteolytic cleavage of PLC-{gamma}1 by caspase may affect tyrosine phosphorylation at Tyr783 and Tyr1254 as well as Tyr771. Since the Tyr771 is adjacent to cleavage site, a defect in the tyrosine phosphorylation at Tyr771 is reasonable. We think that a defect in the tyrosine phosphorylation at other sites such as Tyr783 and Tyr1254 may be due to the conformational change of cleaved PLC-{gamma}1. These results suggest that growth factor-induced tyrosine phosphorylation and apoptosis-induced proteolytic cleavage of PLC-{gamma}1 may reciprocally regulate each other.

Even though several reports have demonstrated that proteolytic cleavage of PLC-{gamma}1 by protease such as V8 protease resulted in the elevation of basal PLC enzyme activity (43) , our results showed that proteolytic cleavage at Asp770 of PLC-{gamma}1 by caspase did not affect its basal enzymatic activity. Contrary to the effect of V8 protease, our results showed that cleaved PLC-{gamma}1 by caspase affect the tyrosine phosphorylation by growth factor receptor kinase, indicating that cleaved PLC-{gamma}1 cannot be activated by growth factor receptor. Consequently, proteolytic cleavage of PLC-{gamma}1 by apoptotic protease seems to cause defective confirmation of PLC-{gamma}1 for activation by growth factor receptor rather than elevation of basal enzymatic activity.

Growth factors including PDGF, EGF, NGF, and IGF-1 induce tyrosine phosphorylation of several signaling molecules and eventually protect cells from apoptosis (18) . Recently, several reports have indicated that there are inactivation mechanisms of growth factor-mediated anti-apoptotic signaling during apoptosis. Among the growth signaling molecules, PKC, PKB/Akt, and Bcl-2 family proteins emerge as central anti-apoptotic proteins. It seems that these anti-apoptotic proteins use phosphorylation as a control mechanism of apoptosis (44 , 45) . In addition, phosphorylation of target protein seems to be the escape mechanism from caspase-dependent cleavage (40) . In a point of apoptosis, apoptotic machinery may need to cleave and inactivate anti-apoptotic proteins for the efficient progression of apoptosis. Widmann et al. (39) have suggested that apoptotic proteases cleave and inactivate several signaling molecules such as Cbl, Raf-1, and Akt-1. Also, caspase-3 converts anti-apoptotic Bcl-2 into a Bax-like death effector by cleaving the loop domain (46) . Protein phosphatases may function as mediators of inactivation of the anti-apoptotic pathway by enhancing cleavage of target proteins by caspases. It has been reported that caspase-3 cleave and activate protein phosphatase 2A during apoptosis (47) .

We have shown that inhibition of PLC activity potentiates apoptosis. Proteolytic cleavage of PLC-{gamma}1 by group II caspases results in the loss of receptor-mediated tyrosine phosphorylation. Tyrosine phosphorylation of PLC-{gamma}1 abrogates its susceptibility to group II caspases. Taken together, it is reasonable to suggest that PLC-{gamma}1 mediates anti-apoptotic function through activation of PKC and that tyrosine phosphorylation of PLC-{gamma}1 by growth factor receptors may provide resistance to caspases. In addition, apoptotic protease may convert unphosphorylated PLC-{gamma}1 into defective PLC-{gamma}1 for the efficient progression of apoptosis. However, a full understanding of the interplay between PLC-{gamma}1 and caspases and their mechanisms based on signal transduction still requires much more study.


   ACKNOWLEDGMENTS
 
We thank Dr. C. B. Chae for critical comments on this manuscript. This work was supported by Korea Science and Engineering Foundation (KOSEF) through the Center for Cell Signaling Research at Ewha Womans University, by KOSEF grant (98–0403-08–01-5), by Bram Korea 21 Program of Minister of Education, and by POSTECH Research Fund (1999).


   FOOTNOTES
 
Received for publication April 23, 1999. Revised for publication December 6, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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