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

Bachelder RE, Ribick MJ, Marchetti A, Falcioni R, Soddu S, Davis KR, Mercurio AM - J. Cell Biol. (1999)

Bottom Line: Biol.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.These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.

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.

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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.
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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.

Mentions: To understand the mechanism by which p53 inhibits α6β4-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 α6β4 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 (Alessi et al. 1996). MDA-MB-435/β4 subclones were transfected with an HA-tagged AKT/PKB construct. These cells were incubated with either a control rat IgG or the β4-specific antibody 439-9B and plated in the absence of serum on secondary antibody–coated tissue culture wells for 1 h. HA immunoprecipitations were performed on extracts from these cells, and the levels of serine-phosphorylated AKT/PKB were assessed by blotting these immunoprecipitates with an antiserum specific for AKT/PKB molecules phosphorylated on serine residue 473. As shown in Fig. 3 A, the antibody-mediated clustering of α6β4 stimulated an increase in the level of serine-phosphorylated AKT/PKB in each of the two MDA-MB-435/β4 subclones relative to control cells (2.1-fold increase, β4 clone 1; 5.5-fold increase, β4 clone 2). This α6β4-induced increase in AKT/PKB serine phosphorylation was dependent on α6β4 signaling based on the inability of α6β4-Δcyt clustering to increase the level of the serine 473–phosphorylated AKT/PKB in MDA-MB-435/β4-Δcyt subclones (data not shown).


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

Bachelder RE, Ribick MJ, Marchetti A, Falcioni R, Soddu S, Davis KR, Mercurio AM - J. Cell Biol. (1999)

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.
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Related In: Results  -  Collection

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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.
Mentions: To understand the mechanism by which p53 inhibits α6β4-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 α6β4 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 (Alessi et al. 1996). MDA-MB-435/β4 subclones were transfected with an HA-tagged AKT/PKB construct. These cells were incubated with either a control rat IgG or the β4-specific antibody 439-9B and plated in the absence of serum on secondary antibody–coated tissue culture wells for 1 h. HA immunoprecipitations were performed on extracts from these cells, and the levels of serine-phosphorylated AKT/PKB were assessed by blotting these immunoprecipitates with an antiserum specific for AKT/PKB molecules phosphorylated on serine residue 473. As shown in Fig. 3 A, the antibody-mediated clustering of α6β4 stimulated an increase in the level of serine-phosphorylated AKT/PKB in each of the two MDA-MB-435/β4 subclones relative to control cells (2.1-fold increase, β4 clone 1; 5.5-fold increase, β4 clone 2). This α6β4-induced increase in AKT/PKB serine phosphorylation was dependent on α6β4 signaling based on the inability of α6β4-Δcyt clustering to increase the level of the serine 473–phosphorylated AKT/PKB in MDA-MB-435/β4-Δcyt subclones (data not shown).

Bottom Line: Biol.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.These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.

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.

Show MeSH
Related in: MedlinePlus