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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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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.
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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.

Mentions: Finally, to demonstrate that the caspase 3–dependent cleavage of AKT/PKB was responsible for the p53 inhibition of AKT/PKB activity in RKO/β4 cells, we explored the effects of a caspase 3 inhibitor on the ability of α6β4 to activate AKT/PKB. HA-AKT/PKB–transfected RKO/β4 cells were subjected to antibody-mediated α6β4 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/PKB–specific rabbit antiserum. As shown in Fig. 7, the pretreatment of RKO/β4 cells with Z-DEVD-FMK restored the ability of α6β4 to stimulate the phosphorylation of AKT/PKB in these cells. These results demonstrate that α6β4 stimulates the caspase 3–dependent cleavage and inactivation of AKT/PKB in p53 wild-type, but not in p53-deficient carcinoma cells.


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)

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.
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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.
Mentions: Finally, to demonstrate that the caspase 3–dependent cleavage of AKT/PKB was responsible for the p53 inhibition of AKT/PKB activity in RKO/β4 cells, we explored the effects of a caspase 3 inhibitor on the ability of α6β4 to activate AKT/PKB. HA-AKT/PKB–transfected RKO/β4 cells were subjected to antibody-mediated α6β4 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/PKB–specific rabbit antiserum. As shown in Fig. 7, the pretreatment of RKO/β4 cells with Z-DEVD-FMK restored the ability of α6β4 to stimulate the phosphorylation of AKT/PKB in these cells. These results demonstrate that α6β4 stimulates the caspase 3–dependent cleavage and inactivation of AKT/PKB in p53 wild-type, but not in p53-deficient carcinoma cells.

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