Correspondence to: Arthur M. Mercurio, Beth Israel Deaconess Medical Center, Research North, 330 Brookline Avenue, Boston, MA 02215. Tel:(617) 667-7714 Fax:(617) 975-5531 E-mail:amercuri{at}bidmc.harvard.edu.
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Abstract |
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Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the 6ß4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:2073320737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of
6ß4 in carcinoma cells that express wild-type p53. In contrast,
6ß4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the
6ß4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of
6ß4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.
Key Words: p53, integrin, AKT/PKB, survival, caspase
PRIMARY epithelial (vß3 (
5ß1 (
6ß1 (
The 6ß4 integrin, a receptor for the laminin family of extracellular matrix proteins, plays an important role in diverse cellular activities. In addition to serving an important structural role in the assembly of hemidesmosomes in epithelial cells (
6ß4 promotes carcinoma cell migration and invasion (
1,000 amino acids (
6ß4 function.
In the present study, we define opposing signaling pathways that are activated by the 6ß4 integrin that promote either carcinoma cell survival or apoptosis, depending on whether these cells express wild-type or functionally inactive mutants of p53. Specifically, we show that
6ß4 can promote the AKT/PKBdependent survival of p53-deficient carcinoma cells. However, this activity contrasts with the ability of
6ß4 to stimulate the caspase-dependent cleavage and inactivation of AKT/PKB in p53 wild-type carcinoma cells. The ability of wild-type p53 to inhibit
6ß4-associated survival signals suggests that the p53 status of an
6ß4-expressing carcinoma cell influences its growth potential.
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Materials and Methods |
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Cells
The RKO colon carcinoma cell line was obtained from M. Brattain (University of Texas, San Antonio, TX), and MDA-MB-435 breast carcinoma cells were obtained from the Lombardi Breast Cancer Depository (Georgetown University).
The cloning of the human ß4 cDNA, the construction of the ß4 cytoplasmic domain deletion mutant (ß4-cyt), and their insertions into the pRc/CMV (ß4) and pcDNA3 (ß4-
cyt) eukaryotic expression vectors, respectively, have been described (
cyt clone 3E1, RKO/ß4 clone D4 (RKO/ß4 clone 1), RKO/ß4 clone A7 (RKO/ß4 clone 2), MDA-MB-435/ß4-
cyt clone 3C12, MDA-MB-435/ß4 clone 5B3 (MDA-MB-435/ß4 clone 1), and MDA-MB-435/ß4 clone 3A7 (MDA/ß4 clone 2) were selected for analysis based on their expression of similar surface levels of
6ß4 and
6ß4-
cyt, as we have previously demonstrated (
Dominant negative p53-expressing RKO/ß4-cyt and RKO/ß4 subclones were obtained by cotransfecting RKO/ß4-
cyt clone 3E1 and RKO/ß4 clone D4 with plasmids expressing the puromycin resistance gene (
cyt cells were transfected with the puromycin resistance gene plasmid alone to obtain puromycin-resistant mock transfectants. All assays were performed using cell maintained below passage 10.
Stable transfectants of MDA/ß4 clone 3A7 that expressed temperature-sensitive p53 were obtained by cotransfecting this cell line with plasmids expressing the puromycin resistance gene (1 mg) (
Dominant negative AKT (dnAKT)/PKBexpressing MDA-MB-435/mock and MDA-MB-435/ß4 transient transfectants were generated by cotransfecting these cell lines using the Lipofectamine reagent (GIBCO BRL) with a plasmid encoding for green fluorescent protein (pEGFP-1; CLONTECH Laboratories; 1 µg) and a dnAKT/PKB construct that contains inactivating mutations in the catalytic domain of AKT/PKB (4 µg) (
Antibodies
The following antibodies were used: 439-9B, a rat mAb specific for the ß4 integrin subunit (
Apoptosis Assays
To induce apoptosis in the RKO and MDA-MB-435 transfectants, the cells were plated in complete medium for 8 h in tissue culture wells (12-well plate; 2.5 x 105 cells/well) that had been coated overnight at 4°C with poly-L-lysine (Sigma Chemical Co.; 2 ml of 25 µg/ml stock) and blocked with 1% BSA. After 8 h, this medium was replaced with serum-free culture medium containing 1% BSA. After 15 h at 37°C, adherent and suspension cells were harvested, combined, and the level of apoptosis in these cells was assessed as described below.
For annexin V stains, cells were washed once with serum-containing medium, once with PBS, once with annexin V-FITC buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and incubated for 15 min at room temperature with annexin V-FITC (Bender MedSystems) at a final concentration of 2.5 µg/ml in annexin V buffer. After washing once with annexin V buffer, the samples were resuspended in the same buffer and analyzed by flow cytometry. Immediately before analysis, propidium iodide was added to a final concentration of 5 µg/ml to distinguish apoptotic from necrotic cells, and 5,000 cells were analyzed for each sample.
For ApopTag reactions, cells were harvested as described above, fixed in 1% paraformaldehyde for 15 min on ice, and washed twice with PBS. The samples were resuspended in 1 ml ice-cold 70% ethanol and stored at -20°C overnight. After centrifugation at 2,500 rpm for 15 min, cells were washed two times in PBS before performing ApopTag reactions (Oncor) according to the manufacturer's recommendations. These samples were analyzed by flow cytometry.
For in situ analysis of apoptosis in cells transfected transiently with the green fluorescent protein (GFP)expressing vector pEGFP-1 (CLONTECH Laboratories) and dnAKT/PKB, the transfected cells were stained with annexin V-PE (PharMingen) according to the manufacturer's directions, and plated on coverslips. The percentage of GFP-positive cells that was annexin V-PEpositive was determined by fluorescence microscopy. A total of at least 80 GFP-positive cells from at least 10 microscopic fields were analyzed for each data point.
Analysis of AKT/PKB Expression and Activity
To assess the expression of endogenous AKT/PKB protein, cells were incubated with either rat Ig or 439-9B as described above in the presence of either DMSO (1:500), a caspase 3 inhibitor (Z-DEVD-FMK; Calbiochem-Novabiochem">Calbiochem-Novabiochem; 4 µg/ml), or a caspase 8 inhibitor (Z-IETD-FMK; Calbiochem-Novabiochem">Calbiochem-Novabiochem; 4 µg/ml). After washing with PBS, the cells were plated in serum-free medium containing 1% BSA in wells of a 12-well plate that had been coated with antirat Ig (13.5 µg/ml) and blocked for 1 h at 37°C with 1% BSA-containing medium. After a 1-h stimulation, adherent and suspension cells were harvested and extracted with AKT/PKB lysis buffer (20 mM Tris, pH 7.4, 0.14 M NaCl, 1% NP-40, 10% glycerol, 2 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml pepstatin, 50 µg/ml leupeptin, 1 mM sodium orthovanadate). After removing cellular debris by centrifugation at 12,000 g for 10 min, equivalent amounts of total cell protein from these extracts were resolved by SDS-PAGE (8%) and transferred to nitrocellulose. The blots were probed with a rabbit anti-AKT/PKB antiserum, followed by HRP-conjugated goat antirabbit Ig, and the immunoreactive bands were visualized by enhanced chemiluminescence. These blots were also probed with a rabbit antiserum specific for actin to confirm the loading of equivalent amounts of protein. Relative AKT/PKB and actin expression levels were assessed by densitometry using IP Lab Spectrum software (Scanalytics).
To determine the level of serine 473phosphorylated AKT/PKB, cells were transfected transiently using the Lipofectamine reagent (GIBCO BRL) with an HA-tagged AKT/PKB cDNA (provided by A. Toker, Boston Biomedical Research Institute, Boston, MA). 20 h after transfection, these cells were harvested by trypsinization and subjected to antibody-mediated integrin clustering. Specifically, cells were incubated on ice for 30 min with either control rat IgG or 439-9B at a concentration of 10 µg/ml. After washing with PBS, the cells were plated in serum-free medium containing 1% BSA onto wells of a 60-mM tissue culture dish that had been coated at 4°C with antirat Ig (13.5 µg/ml) and blocked for 1 h at 37°C in 1% BSA-containing medium. After 1 h, adherent and suspension cells were harvested and washed twice with PBS. Proteins from these cells were extracted with AKT/PKB lysis buffer (see above). After removing cellular debris by centrifugation at 12,000 g for 10 min at 4°C, equivalent amounts of total cellular protein were precleared with a 1:1 mixture of protein A and protein GSepharose for 1 h at 4°C. Immunoprecipitations were performed for 1 h on these precleared lysates using an HA-specific mAb (1 µg; Boehringer Mannheim) and protein A/protein GSepharose beads. Proteins from these immunoprecipitates were subjected to reducing SDS-PAGE (8%), transferred to nitrocellulose, and probed with an AKT/PKB phosphoserine 473specific rabbit antiserum (New England Biolabs) followed by HRP-conjugated goat antirabbit IgG. Phospho-AKT/PKB was detected on these blots by chemiluminescence (Pierce Chemical Co.). These samples were also probed with rabbit anti-AKT/PKB. The relative intensity of phosphoserine AKT/PKB and AKT/PKB bands was assessed by densitometry, as described above.
Analysis of AKT/PKB Proteolysis
Baculovirus-expressed AKT/PKB (0.5 µg; provided by A. Toker) was incubated with either active recombinant caspase 8 (2 mg; Calbiochem-Novabiochem">Calbiochem-Novabiochem) or active recombinant caspase 3 (2 µg; Calbiochem-Novabiochem">Calbiochem-Novabiochem) at 37°C for 1 h in a final volume of 10 µl. Subsequently, the reaction mixtures were divided into two aliquots and resolved by SDS-PAGE (8%). The gels were silver stained using the GelCode SilverSNAP Stain Kit (Pierce Chemical Co.) or transferred to nitrocellulose and probed with a rabbit AKT/PKB antiserum as described above.
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Results |
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The 6ß4 Integrin Promotes the Survival of p53-deficient, but Not p53 Wild-type Carcinoma Cells
For our initial experiments, we used stable ß4 transfectants of two 6ß4-deficient carcinoma cell lines that differ in their p53 status: RKO colon carcinoma cells, which express wild-type p53 (
6ß4 (RKO/ß4-
cyt; MDA/b4-
cyt) that is signaling deficient. The characterization of these cells has been described previously (
To explore the potential influence of 6ß4 expression on the survival of serum-starved carcinoma cells deprived of matrix attachment, the
6ß4 and
6ß4-
cytexpressing RKO and MDA-MB-435 subclones were plated on poly-L-lysine in serum-free medium. The level of apoptosis in these populations was determined either by staining with annexin V-FITC to detect cells in the early stages of apoptosis or by performing terminal deoxynucleotidyl transferase end labeling reactions (Apoptag) to detect DNA fragmentation (Fig 1). In addition, we assessed the viability of these serum-deprived cells by measuring the cellular uptake of propidium iodide (Table 1). The ability of
6ß4 to promote the survival of these cells was determined by subtracting the percent apoptotic
6ß4-expressing cells from the percent apoptotic
6ß4-
cytexpressing cells. The expression of
6ß4 in MDA-MB-435 cells significantly increased the survival of these cells relative to MDA-MB-435 cells expressing
6ß4-
cyt, as assessed by annexin V-FITC staining (Fig 1), ApopTag staining (Fig 1), and propidium iodide uptake (Table 1). In contrast, the expression of
6ß4 in RKO cells did not increase the survival of these cells relative to either the mock (Table 1) or RKO/ß4-
cyt transfectants (Fig 1). In fact, we observed a higher level of apoptosis and cell death in serum-starved RKO/ß4 as compared with RKO/ß4-
cyt cells, in agreement with our previous demonstration that
6ß4 can promote apoptosis in wild-type p53 carcinoma cells (
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Based on the fact that RKO and MDA-MB-435 cells differ in their p53 status, we reasoned that the ability of 6ß4 to promote cell survival may be inhibited by p53. This hypothesis was examined by investigating the effect of
6ß4 expression on the survival of RKO cells in which p53 activity had been inhibited by the expression of a dnp53 construct. Indeed,
6ß4 expression promoted the survival of serum-starved, dnp53-expressing RKO cells as determined by ApopTag and annexin V-FITC staining (Fig 1). These results demonstrate that p53 can suppress the survival signaling mediated by
6ß4 in serum-starved carcinoma cells.
6ß4-Mediated Survival in p53-deficient Carcinoma Cells Is Inhibited by Dominant Negative AKT/PKB
Given the importance of the AKT/PKB kinase in numerous survival signaling pathways (6ß4 in serum-starved, p53-deficient carcinoma cells was AKT/PKBdependent. The MDA-MB-435/ß4transfected clones, as well as the parental cells, were cotransfected with plasmids encoding for GFP and an HA-tagged, kinase-deficient AKT/PKB mutant that acts as a dominant negative construct (dnAKT/PKB) (
6ß4 survival function in each of the two MDA-MB-435/ß4 clones examined, but it did not alter the level of apoptosis in parental MDA-MB-435 cells.
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p53 Inhibits the Activation of AKT/PKB by 6ß4
To understand the mechanism by which p53 inhibits 6ß4-mediated survival, we investigated the possibility that p53 alters the ability of this integrin to activate AKT/PKB. Initially, we examined whether the antibody-mediated clustering of
6ß4 in MDA-MB-435 cells resulted in the phosphorylation of AKT/PKB on serine 473, an event that has been shown to correlate with AKT/PKB activation (
6ß4 stimulated an increase in the level of serine-phosphorylated AKT/PKB in each of the two MDA-MB-435/ß4 subclones relative to control cells (2.1-fold increase, ß4 clone 1; 5.5-fold increase, ß4 clone 2). This
6ß4-induced increase in AKT/PKB serine phosphorylation was dependent on
6ß4 signaling based on the inability of
6ß4-
cyt clustering to increase the level of the serine 473phosphorylated AKT/PKB in MDA-MB-435/ß4-
cyt subclones (data not shown).
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To investigate the influence of p53 on the activation of AKT/PKB by 6ß4, we explored whether
6ß4 clustering induced the phosphorylation of AKT/PKB on serine residue 473 in MDA-MB-435/ß4 that had been reconstituted with functional p53. Specifically, MDA-MB-435/ß4 cells were transfected with a temperature-sensitive mutant of human p53 (tsp53) that assumes a functional conformation at 32°C but not at 37°C (
6ß4 significantly increased the level of phosphoserine 473-AKT/PKB in mock-transfected MDA/ß4 cells (7.9-fold increase), but not in tsp53-expressing MDA/ß4 cells (1.2-fold increase). The importance of p53 in the inhibition of the
6ß4-associated activation of AKT/PKB was indicated by the finding that
6ß4 clustering increased the level of phosphoserine 473 AKT/PKB in MDA/ß4 + tsp53 transfectants that had been incubated at 37°C, the nonpermissive temperature for this tsp53 construct (data not shown).
The ability of p53 to suppress the 6ß4-mediated activation of AKT/PKB was explored further in RKO carcinoma cells, which express wild-type p53. In agreement with the results obtained in MDA/ß4 cells that had been reconstituted with functional p53, the clustering of
6ß4 in two independent RKO/ß4 subclones did not result in increased amounts of serine phosphorylated AKT/PKB (Fig 3 C and data not shown). Importantly, the expression of dnp53 in RKO/ß4 cells restored the ability of
6ß4 to activate AKT/PKB, as evidenced by an increase in phosphoserine 473-AKT/PKB immunoreactivity in RKO/ß4 + dnp53 cells that had been subjected to antibody-mediated
6ß4 clustering (8.6-fold increase), as described above (Fig 3 C). The ability of
6ß4 to stimulate AKT/PKB activity in RKO/ß4 + dnp53 cells but not in RKO/ß4 cells was confirmed by performing in vitro kinase assays using histone H2B as a substrate (data not shown). As a control for specificity, we also demonstrated that the clustering of
6ß4 on dnp53-expressing RKO/ß4-
cyt cells did not stimulate AKT/PKB activity (data not shown).
6ß4 Stimulation Induces the Caspase 3dependent Cleavage of AKT/PKB in a p53-dependent Manner
To define the mechanism by which p53 inhibits the ability of 6ß4 to activate AKT/PKB, we investigated whether p53 alters AKT/PKB expression levels in response to
6ß4 clustering. RKO/ß4 and RKO/ß4 + dnp53-expressing cells were incubated with either rat Ig or 439-9B and stimulated on secondary antibodycoated wells for 1 h. The amount of total AKT/PKB in equivalent amounts of total protein from these lysates was assessed by immunoblotting. Importantly, the antibody-mediated clustering of the
6ß4 integrin on each of two RKO/ß4 subclones resulted in a significant reduction in the total level of AKT/PKB in these cells (Fig 4 A). In contrast, AKT/PKB levels were not reduced in dnp53-expressing RKO/ß4 cells (Fig 4 B) or in MDA-MB-435/ß4 subclones (data not shown) after the antibody-mediated clustering of
6ß4. We also observed decreased levels of HA-AKT/PKB protein in HA-AKT/PKBtransfected RKO/ß4 cells, but not in HA-AKT/PKBtransfected RKO/ß4 + dnp53 cells upon the antibody-mediated clustering of
6ß4 (data not shown).
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Based on the reported ability of caspases to cleave signaling molecules that promote cell survival (6ß4 may promote the caspase-dependent cleavage of AKT/PKB in wild-type p53-expressing carcinoma cells. Initially, we explored the importance of caspase 3 activity, which has been shown to play a crucial role in p53-dependent apoptotic pathways (
6ß4-associated reduction of AKT/PKB expression levels. In agreement with the data shown in Fig 4, the clustering of
6ß4 in control RKO/ß4 cells significantly reduced the level of AKT/PKB in these carcinoma cells (Fig 5). However, RKO/ß4 cells that had been pretreated with Z-DEVD-FMK, a cell permeable caspase 3 inhibitor, did not exhibit decreased levels of AKT/PKB in response to
6ß4 clustering (Fig 5). In contrast, we detected a decreased amount of AKT/PKB after the clustering of
6ß4 in RKO/ß4 cells that had been pretreated with Z-IETD-FMK, a cell permeable caspase 8 inhibitor (Fig 5). Importantly, no effect of these inhibitors on AKT/PKB levels was observed upon the clustering of
6ß4 on RKO/
6ß4-
cyt cells (data not shown).
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The ability of the caspase 3 inhibitor to restore normal AKT/PKB levels suggested that AKT/PKB is cleaved by caspase 3 upon the clustering of 6ß4 in carcinoma cells expressing wild-type p53. To establish the caspase 3mediated cleavage of AKT/PKB more rigorously, we investigated whether a recombinant form of this cysteine protease could cleave baculovirus-expressed AKT/PKB in vitro. Proteins in these reactions were resolved by SDS-PAGE and detected by silver staining. The results obtained revealed that the incubation of baculovirus-expressed AKT/PKB (Mr, 60 kD) with recombinant caspase 3 resulted in the formation of an AKT/PKB cleavage product (Mr, 49 kD) (Fig 6). In contrast, we did not detect an AKT/PKB cleavage product after the incubation of baculovirus AKT/PKB with recombinant caspase 8 (Fig 6). The caspase 3generated AKT/PKB cleavage product was also detected by immunoblotting with an antiserum specific for the carboxy terminus of AKT/PKB, suggesting that caspase 3 cleaves AKT/PKB at its amino terminus (data not shown).
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Finally, to demonstrate that the caspase 3dependent cleavage of AKT/PKB was responsible for the p53 inhibition of AKT/PKB activity in RKO/ß4 cells, we explored the effects of a caspase 3 inhibitor on the ability of 6ß4 to activate AKT/PKB. HA-AKT/PKBtransfected RKO/ß4 cells were subjected to antibody-mediated
6ß4 clustering in the presence of either DMSO or the caspase 3 inhibitor Z-DEVD-FMK. HA immunoprecipitates from extracts from these cells were subjected to immunoblotting with the phosphoserine 473 AKT/PKBspecific rabbit antiserum. As shown in Fig 7, the pretreatment of RKO/ß4 cells with Z-DEVD-FMK restored the ability of
6ß4 to stimulate the phosphorylation of AKT/PKB in these cells. These results demonstrate that
6ß4 stimulates the caspase 3dependent cleavage and inactivation of AKT/PKB in p53 wild-type, but not in p53-deficient carcinoma cells.
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Discussion |
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The binding of extracellular matrix proteins to integrins initiates survival signals that inhibit anoikis, a form of apoptosis induced upon the detachment of cells from extracellular matrix (6ß4 integrin suppresses anoikis exclusively in carcinoma cells that lack functional p53. Furthermore, we demonstrate that this
6ß4-associated survival function depends on the ability of this integrin to activate the serine/threonine kinase AKT/PKB in p53-deficient cells. Finally, we provide evidence that p53 inhibits the
6ß4-mediated activation of AKT/PKB by promoting the caspase 3dependent cleavage of this kinase. Collectively, our findings establish that p53 can inhibit an integrin-associated survival function, a phenomenon that has important implications for tumor cell growth.
Our results suggest that the 6ß4 integrin can enhance the survival of carcinoma cells in an AKT/PKBdependent manner. Although previous studies have shown that cell attachment to matrix proteins promotes the survival of primary epithelial cells (
6ß4 is the first specific integrin to be implicated in the delivery of AKT/PKBdependent survival signals to carcinoma cells. The importance of AKT/PKB in
6ß4 survival signaling was indicated in our studies by the ability of a dnAKT/PKB construct containing inactivating mutations in the catalytic domain to inhibit the survival effect of
6ß4 in serum-starved MDA-MB-435 cells. Although this dnAKT/PKB has been used extensively to implicate AKT/PKB in survival pathways, it is possible that it associates with phosphoinositide-dependent kinases and inhibits their activity. However, our observation that the expression of a constitutively active AKT/PKB in MDA-MBA-435 enhances their survival (data not shown) strongly suggests that
6ß4 expression promotes the survival of these cells by activating AKT/PKB.
Our demonstration that p53 can inhibit AKT/PKB kinase activity is of interest in light of the recent finding that the PTEN tumor suppressor can also inhibit cell growth by inhibiting AKT/PKB in a manner that is dependent on its lipid phosphatase activity (6ß4 survival signaling by promoting the caspase-dependent cleavage of AKT/PKB provides a mechanistic link between tumor suppressor function and the regulation of integrin signaling, similar to the phosphatase activities of PTEN. Although previous studies have demonstrated that caspases can be activated by p53 in both cell-free systems (
The finding that AKT/PKB activity can be regulated by caspase 3 substantiates the hypothesis that caspases play an important role in many forms of apoptosis based on their ability to cleave signaling molecules that influence cell survival. For example, caspases have been shown to cleave and inactivate an inhibitor of caspase-activated deoxyribonuclease (CAD). Importantly, the cleavage of this inhibitor results in the activation of CAD, which is the enzyme responsible for the DNA fragmentation that is characteristic of apoptosis (Enari et al., 1998; 6ß4 integrin survival function.
It is important to consider the mechanism by which the 6ß4-induced, caspase-dependent cleavage of AKT/PKB inhibits its kinase activity. We detected an AKT/PKB fragment (Mr, 49 kD) after the in vitro incubation of AKT/PKB with recombinant caspase 3. This fragment was recognized by a rabbit antiserum raised against a peptide corresponding to the extreme carboxy-terminal amino acids of the molecule, suggesting that caspase 3 cleaves AKT/PKB at its amino terminus. Interestingly, the pleckstrin homology domain, which resides in the amino terminus of AKT/PKB, is important in both the translocation of this kinase to the membrane and its subsequent activation (
6ß4, despite our detection of reduced AKT/PKB levels under these conditions. This result suggests that after the initial cleavage of AKT/PKB by caspase 3, this kinase is subjected to further cleavage by other caspases, as has been shown for ICAD (
6ß4 suggests that AKT/PKB cannot be detected by immunoblotting after its cleavage by multiple caspases. The ability of a caspase 3 inhibitor to restore both normal AKT/PKB levels as well as the
6ß4-mediated activation of AKT/PKB suggests that the degradation of AKT/PKB observed in vivo is dependent on the initial cleavage of this kinase by caspase 3.
In contrast to our finding that p53-dependent, caspase 3 activity inhibits AKT/PKB, other studies have concluded that constitutively active AKT/PKB can delay p53-dependent apoptosis (6ß4 clustering to promote the caspase 3dependent inactivation of AKT/PKB in p53 wild-type carcinoma cells may relate to the fact that
6ß4 signaling stimulates caspase activity before AKT/PKB activity in these cells. Alternatively, it is possible that the ability of caspase 3 to cleave AKT/PKB was not observed in previous studies because insufficient amounts of endogenous caspase activity were present to inhibit the activity of exogenously introduced, active AKT/PKB. Nonetheless, these results suggest that an intimate crosstalk exists between AKT/PKB and caspases that contributes to the regulation of cell survival.
We have previously demonstrated that the 6ß4 integrin activates p53 function (
6ß4 activity, namely the inhibition of AKT/PKB activity and its associated cell survival function. Similar to previous results from our laboratory (
6ß4 is ligand-independent in ß4-transfected, p53-deficient carcinoma cells. This ligand-independent survival function may be attributable to the ability of the ß4 cytoplasmic domain to self-associate (
In addition to demonstrating that p53 inhibits 6ß4-mediated survival, we observed that
6ß4 increases the level of apoptosis observed in serum-starved p53 wild-type carcinoma cells. This result suggests that the apoptotic signaling pathway activated by
6ß4 can augment the apoptotic signaling initiated by serum deprivation. Although p53 has been implicated in the apoptosis induced in endothelial cells upon their detachment from matrix (
6ß4 apoptotic signaling requires p53 activity (
6ß4 apoptotic signaling.
The current studies may explain why the 6ß4 integrin has been implicated in the apoptosis of some cells and the survival of others. Specifically,
6ß4 has been shown to induce growth arrest and apoptosis in several carcinoma cell lines (
6ß4 may relate to the fact that the functions of
6ß4 are cell typespecific. The current studies establish that the p53 tumor suppressor is one critical signaling molecule that may influence
6ß4 function in different cell types because this integrin promotes apoptosis only in wild-type p53-expressing cells and survival only in p53-deficient cells. Interestingly, the reported ability of
6ß4 to promote keratinocyte survival (
One implication of our findings is that the 6ß4 integrin is similar to a number of oncogenes that promote cell proliferation in some settings and cell death in others. The recent observation that oncogenes can deliver such death signals has led to their seemingly contradictory categorization as tumor suppressors in select environments. For example, although the stimulation of c-myc and E2F normally promotes cell proliferation, the activation of these oncogenes induces apoptosis in the presence of secondary stress signals such as p53 expression, serum starvation or hypoxia (
6ß4 integrin, which promotes the survival of p53-deficient cells, could also be classified loosely as a tumor suppressor based on its apoptotic function in carcinoma cells that express wild-type p53. The current studies demonstrate that, similar to the activity of oncogenes, integrin function and signaling can be profoundly influenced by physiological stimuli that activate other signaling pathways in a cell.
In summary, we have described the ability of the 6ß4 integrin to promote the survival of the p53 mutant, but not p53 wild-type carcinoma cells. This ability of p53 to influence integrin-mediated functions so markedly derives from its ability to activate the caspase 3dependent cleavage of AKT/PKB. The fact that AKT/PKB overexpression has been suggested to contribute to the transformed phenotype of tumor cells (
6ß4 integrin into p53 wild-type tumors may inhibit their growth by inducing the cleavage of this transforming protein. The ability of
6ß4 to induce the p53-dependent cleavage of AKT/PKB also suggests that the acquisition of inactivating mutations in either p53 or caspase 3 will provide a selective growth advantage for carcinoma cells by stimulating
6ß4-mediated AKT/PKBdependent survival signaling. Moreover, given our previous demonstration that
6ß4 promotes carcinoma cell migration and invasion (
6ß4 and mutant forms of p53 or caspase 3 will have a distinct advantage in their ability to disseminate and survive as metastatic lesions.
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Footnotes |
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1 Abbreviations used in this paper: CAD, caspase-activated deoxyribonuclease; dnAKT, dominant negative AKT; dnp53, dominant negative p53; GFP, green fluorescent protein; HA, hemagglutinin; tsp53, temperature-sensitive p53.
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Acknowledgements |
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We thank Moshe Oren, Alt Zantema, Alex Toker, and Phil Hinds (Harvard Medical School, Boston, MA) for reagents. We also thank Lewis Cantley, Alex Toker, Phil Hinds, Kathy O'Connor, and Leslie Shaw (Beth Israel Deaconess Medical Center, Boston, MA) for valuable discussions.
This work was supported by National Institutes of Health grants CA80789, AI39264 (both to A.M. Mercurio), and CA81697 (to R.E. Bachelder), as well as by the Italian Association for Cancer Research.
Submitted: 14 May 1999
Revised: 13 October 1999
Accepted: 18 October 1999
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References |
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