p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB

Abstract

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 alpha6beta4 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. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 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 alpha6beta4-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 alpha6beta4 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.

Figure 1

p53 inhibits α6β4-mediated survival. MDA-MB-435, RKO, and RKO + dnp53 cells that expressed either α6β4 (β4) or α6β4-Δcyt (β4-Δcyt) were plated on poly-l-lysine–coated tissue culture wells and cultured in the absence of serum. After 15 h, the cells were harvested, subjected to either ApopTag reactions (A) or annexin V-FITC staining (B), and analyzed by flow cytometry. A survival effect of α6β4 was quantified by subtracting the percentage of α6β4-expressing cells that were positive for either Apoptag (A) or annexin V-FITC (B) staining from the percentage of α6β4-Δcyt–expressing cells that were positive for these markers. This value was plotted on the bar graphs shown in A and B, with positive values indicating that the specified β4 clone exhibits increased survival relative to the relevant β4-Δcyt subclone, and negative values indicating an increased apoptosis of the indicated clone relative to the appropriate β4-Δcyt clone. The data in A represent the means (± SEM) from three independent experiments. Similar results to those shown in B were observed in three separate trials.

Figure 2

Expression of a dominant negative AKT/PKB inhibits α6β4-mediated survival. Parental (neo) and α6β4-expressing (β4) MDA-MB-435 cells were transfected with either a GFP-expressing plasmid (mock) or both a GFP and a dnAKT/PKB–expressing construct (dnAKT/PKB), plated on poly-l-lysine, and cultured for 15 h in the absence of serum. Apoptosis in these cells was assessed by annexin V-PE staining. The data are reported as the percentage of GFP-positive cells that were stained by annexin V-PE. Similar results were observed in two additional experiments.

Figure 3

p53 inhibits the ability of α6β4 to induce AKT/PKB phosphorylation in carcinoma cells. MDA/β4, MDA/β4 + tsp53, RKO/β4, and RKO/β4 + dnp53 cells were transfected transiently with an HA-tagged AKT/PKB. These transfectants were incubated with the indicated primary antibodies, washed, and plated in the absence of serum on secondary antibody–coated tissue culture wells. HA-AKT/PKB–transfected MDA/β4 (A), RKO/β4 (C), and RKO/β4 + dnp53 (C) cells were stimulated for 1 h at 37°C. Alternatively, mock- and tsp53-transfected MDA/β4 cells (B) were stimulated for 1 h at 32°C to activate tsp53, followed by an additional hour at 37°C to activate AKT/PKB. Immunoprecipitations were performed with an HA-specific mAb on equal amounts of total extracted protein. The immunoprecipitates were resolved by SDS-PAGE (8%), transferred to nitrocellulose, and probed with a phosphoserine 473 AKT/PKB–specific rabbit antiserum (New England Biolabs), followed by HRP-conjugated goat anti–rabbit IgG. Phosphoserine-specific AKT/PKB bands were detected by chemiluminescence, and are noted by arrows.

Figure 4

Clustering of the α6β4 integrin reduces AKT/PKB protein levels in p53-wild type but not in p53-deficient carcinoma cells. RKO/β4 (A and B) and RKO/β4 + dnp53 (B)–expressing cells were incubated with either rat Ig or 439-9B and plated on secondary antibody–coated wells for 1 h in the absence of serum. Equivalent amounts of total protein from lysates from these cells were resolved by SDS-PAGE (8%), transferred to nitrocellulose, and probed with an AKT/PKB–specific rabbit antiserum (New England Biolabs) followed by HRP-conjugated goat anti–rabbit IgG. These blots were also probed with an actin-specific rabbit antiserum (Sigma Chemical Co.) to confirm the loading of equivalent amounts of protein. The AKT/PKB and actin bands were detected by enhanced chemiluminescence, and are indicated by arrows. These bands were quantified by densitometry. α6β4 clustering decreased AKT/PKB levels in RKO/β4 subclones (1.7-fold decrease, β4 clone 1; 1.9-fold decrease, β4 clone 2), but not in RKO/β4 + dnp53 cells. Similar results were observed in four additional trials.

Figure 5

A caspase 3 inhibitor blocks α6β4-associated reductions in AKT/PKB protein levels. RKO/β4 cells were incubated with either rat Ig or 439-9B in the presence of DMSO (1:500), a caspase 3 inhibitor (Z-DEVD-FMK; 4 μg/ml), or a caspase 8 inhibitor (Z-IETD-FMK; 4 μg/ml). These cells were washed with PBS and plated onto secondary antibody–coated wells in the presence of the same drugs for 1 h in serum-free medium. Equivalent amounts of total protein were resolved by SDS-PAGE (8%), transferred to nitrocellulose, and probed with an AKT/PKB–specific rabbit antiserum (New England Biolabs) followed by HRP-conjugated goat anti–rabbit IgG. AKT/PKB was detected by enhanced chemiluminescence and quantified by densitometry. The antibody-mediated clustering of α6β4 decreased the level of AKT/PKB in DMSO-treated cells (2.0-fold decrease, β4 clone 1; 1.9-fold decrease, β4 clone 2), as well as in cells pretreated with a caspase 8 inhibitor (1.9-fold decrease). In contrast, the pretreatment of these cells with a caspase 3 inhibitor partially restored AKT/PKB levels in RKO/β4 cells subjected to α6β4 clustering (1.1-fold decrease, β4 clone 1; 1.1-fold decrease, β4 clone 2). By probing these blots with an actin-specific rabbit antiserum (Sigma Chemical Co.), we confirmed that equivalent amounts of actin were present in each lane (data not shown). Similar results were observed in three experiments.

Figure 6

AKT/PKB is cleaved by recombinant caspase 3 in vitro. Baculovirus-expressed AKT/PKB (0.5 μg) was incubated either alone, with recombinant caspase 3 (2 μg) or with recombinant caspase 8 (2 μg) for 1 h at 37°C. Proteins in these reactions were resolved by SDS-PAGE (8%) and subjected to silver staining. AKT/PKB and its cleavage product are indicated by arrows. Similar results were observed in three trials.

Figure 7

A caspase 3 inhibitor restores the ability of α6β4 to induce AKT/PKB phosphorylation. HA-AKT/PKB–transfected RKO/β4 cells were incubated with either rat Ig or 439-9B in the presence of DMSO (1:500) or a caspase 3 inhibitor (Z-DEVD-FMK; 4 μg/ml). After washing with PBS, these cells were plated on secondary antibody–coated wells in serum-free medium containing the indicated drugs for 1 h. HA immunoprecipitations were performed on equivalent amounts of total extracted protein from these samples. These immunoprecipitates were resolved by SDS-PAGE (8%), transferred to nitrocellulose, and probed with rabbit antiserum specific for phosphoserine 473-AKT/PKB, followed by HRP-conjugated goat anti–rabbit Ig. Phosphoserine 473-AKT/PKB was detected by enhanced chemiluminescence, and is indicated by an arrow. Total AKT/PKB levels were also assessed by stripping these membranes and probing with an AKT/PKB–specific rabbit antiserum (data not shown). Relative activity was assessed by determining the ratio of serine phosphorylated AKT/PKB to that of total AKT/PKB for each sample (relative AKT activity: lane 1 = 1.0; lane 2 = 1.3; lane 3 = 1.1; and lane 4 = 3.1). Similar results were observed in three experiments.