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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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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.
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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.

Mentions: Given the importance of the AKT/PKB kinase in numerous survival signaling pathways (Ahmed et al. 1997; Datta et al. 1997; Dudek et al. 1997; Songyang et al. 1997; Blume-Jensen et al. 1998; Crowder and Freeman 1998; Gerber et al. 1998), we investigated whether the survival function of α6β4 in serum-starved, p53-deficient carcinoma cells was AKT/PKB–dependent. The MDA-MB-435/β4–transfected 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) (Dudek et al. 1997; Skorski et al. 1997; Eves et al. 1998). Expression of this dnAKT/PKB construct was confirmed by immunoblotting extracts from these transfected cells with an HA-specific mAb (data not shown). After 15 h of serum starvation, the level of apoptosis in GFP-positive cells was assessed by annexin V-PE staining. As shown in Fig. 2, MDA-MB-435/β4 clones demonstrated significantly less apoptosis than parental MDA-MB-435 cells in agreement with the data shown in Table . Importantly, dnAKT/PKB expression inhibited this α6β4 survival function in each of the two MDA-MB-435/β4 clones examined, but it did not alter the level of apoptosis in parental MDA-MB-435 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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169339&req=5

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.
Mentions: Given the importance of the AKT/PKB kinase in numerous survival signaling pathways (Ahmed et al. 1997; Datta et al. 1997; Dudek et al. 1997; Songyang et al. 1997; Blume-Jensen et al. 1998; Crowder and Freeman 1998; Gerber et al. 1998), we investigated whether the survival function of α6β4 in serum-starved, p53-deficient carcinoma cells was AKT/PKB–dependent. The MDA-MB-435/β4–transfected 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) (Dudek et al. 1997; Skorski et al. 1997; Eves et al. 1998). Expression of this dnAKT/PKB construct was confirmed by immunoblotting extracts from these transfected cells with an HA-specific mAb (data not shown). After 15 h of serum starvation, the level of apoptosis in GFP-positive cells was assessed by annexin V-PE staining. As shown in Fig. 2, MDA-MB-435/β4 clones demonstrated significantly less apoptosis than parental MDA-MB-435 cells in agreement with the data shown in Table . Importantly, dnAKT/PKB expression inhibited this α6β4 survival function in each of the two MDA-MB-435/β4 clones examined, but it did not alter the level of apoptosis in parental MDA-MB-435 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