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CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt.

Fukuda T, Guo L, Shi X, Wu C - J. Cell Biol. (2003)

Bottom Line: Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2.However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation.Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

ABSTRACT
Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

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CH-ILKBP is crucial for cell survival. (A) HeLa cells, the control transfectants, and the CH-ILKBP siRNA transfectants were fixed and then incubated in the TUNEL reaction mixture, washed, and stained with DAPI. Apoptotic (TUNEL-positive) cells were detected by fluorescence microscopy. (B) The percentages of TUNEL-positive cells were calculated by counting at least 800 cells from five randomly selected fields. Data represent means ± SD. (C) The activities of caspase-3 in HeLa cells and the control and CH-ILKBP siRNA transfectants were quantified. Data represent means ± SD from two independent experiments.
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fig2: CH-ILKBP is crucial for cell survival. (A) HeLa cells, the control transfectants, and the CH-ILKBP siRNA transfectants were fixed and then incubated in the TUNEL reaction mixture, washed, and stained with DAPI. Apoptotic (TUNEL-positive) cells were detected by fluorescence microscopy. (B) The percentages of TUNEL-positive cells were calculated by counting at least 800 cells from five randomly selected fields. Data represent means ± SD. (C) The activities of caspase-3 in HeLa cells and the control and CH-ILKBP siRNA transfectants were quantified. Data represent means ± SD from two independent experiments.

Mentions: We employed RNAi to deplete CH-ILKBP in human cells. Transfection of HeLa cells with a CH-ILKBP small interfering RNA (siRNA) (Fig. 1 A, lane 1) but not an irrelevant control RNA (Fig. 1 A, lane 2) resulted in a near total loss of CH-ILKBP. Probing the same samples with an antiactin antibody showed that the level of actin was not altered (Fig. 1 B). Thus, the CH-ILKBP siRNA effectively suppresses the expression of CH-ILKBP without globally altering protein expression. CH-ILKBP forms a ternary complex with ILK and PINCH-1 in cells (Tu et al., 2001). Consistent with this, depletion of CH-ILKBP modestly reduced the levels of ILK (Fig. 1 C) and PINCH-1 (Fig. 1 D). The suppression of CH-ILKBP expression in the CH-ILKBP siRNA transfectants (Fig. 1 E) but not the control siRNA transfectants (Fig. 1 F) was confirmed by immunofluorescent staining. Staining of the cells with an antibody to paxillin (a marker of focal adhesions) showed that focal adhesions were formed in the CH-ILKBP–deficient cells (Fig. 1 G) and the control cells (Fig. 1 H). Moreover, staining of the cells with an anti-ILK antibody showed that a substantial amount of ILK (or proteins that share a common epitope with ILK) was clustered at focal adhesions (Fig. 1 I) despite the suppression of CH-ILKBP expression. To further analyze this, we transfected HeLa cells expressing GFP-ILK or GFP–PINCH-1 with CH-ILKBP siRNA and the control RNA, respectively. GFP-ILK (Fig. 1 L) and GFP–PINCH-1 (Fig. 1 N) localized to cell–matrix adhesions in both the CH-ILKBP siRNA transfectants (Fig. 1, K and M) and the control transfectants (unpublished data). Although depletion of CH-ILKBP prevented neither the formation of cell–matrix adhesions nor the localization of ILK and PINCH-1 to the adhesion sites, it induced a dramatic increase of apoptosis as revealed by the appearance of a large percentage of TUNEL-positive cells (Fig. 2, A and B). Analyses of the activity of caspase-3, a key mediator of apoptosis, showed that its activity was markedly increased in the absence of CH-ILKBP (Fig. 2 C). These results suggest that (a) CH-ILKBP is essential for cell survival albeit it is not absolutely required for the formation of cell–matrix adhesions and (b) CH-ILKBP acts in the cell survival pathway upstream of caspase-3.


CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt.

Fukuda T, Guo L, Shi X, Wu C - J. Cell Biol. (2003)

CH-ILKBP is crucial for cell survival. (A) HeLa cells, the control transfectants, and the CH-ILKBP siRNA transfectants were fixed and then incubated in the TUNEL reaction mixture, washed, and stained with DAPI. Apoptotic (TUNEL-positive) cells were detected by fluorescence microscopy. (B) The percentages of TUNEL-positive cells were calculated by counting at least 800 cells from five randomly selected fields. Data represent means ± SD. (C) The activities of caspase-3 in HeLa cells and the control and CH-ILKBP siRNA transfectants were quantified. Data represent means ± SD from two independent experiments.
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fig2: CH-ILKBP is crucial for cell survival. (A) HeLa cells, the control transfectants, and the CH-ILKBP siRNA transfectants were fixed and then incubated in the TUNEL reaction mixture, washed, and stained with DAPI. Apoptotic (TUNEL-positive) cells were detected by fluorescence microscopy. (B) The percentages of TUNEL-positive cells were calculated by counting at least 800 cells from five randomly selected fields. Data represent means ± SD. (C) The activities of caspase-3 in HeLa cells and the control and CH-ILKBP siRNA transfectants were quantified. Data represent means ± SD from two independent experiments.
Mentions: We employed RNAi to deplete CH-ILKBP in human cells. Transfection of HeLa cells with a CH-ILKBP small interfering RNA (siRNA) (Fig. 1 A, lane 1) but not an irrelevant control RNA (Fig. 1 A, lane 2) resulted in a near total loss of CH-ILKBP. Probing the same samples with an antiactin antibody showed that the level of actin was not altered (Fig. 1 B). Thus, the CH-ILKBP siRNA effectively suppresses the expression of CH-ILKBP without globally altering protein expression. CH-ILKBP forms a ternary complex with ILK and PINCH-1 in cells (Tu et al., 2001). Consistent with this, depletion of CH-ILKBP modestly reduced the levels of ILK (Fig. 1 C) and PINCH-1 (Fig. 1 D). The suppression of CH-ILKBP expression in the CH-ILKBP siRNA transfectants (Fig. 1 E) but not the control siRNA transfectants (Fig. 1 F) was confirmed by immunofluorescent staining. Staining of the cells with an antibody to paxillin (a marker of focal adhesions) showed that focal adhesions were formed in the CH-ILKBP–deficient cells (Fig. 1 G) and the control cells (Fig. 1 H). Moreover, staining of the cells with an anti-ILK antibody showed that a substantial amount of ILK (or proteins that share a common epitope with ILK) was clustered at focal adhesions (Fig. 1 I) despite the suppression of CH-ILKBP expression. To further analyze this, we transfected HeLa cells expressing GFP-ILK or GFP–PINCH-1 with CH-ILKBP siRNA and the control RNA, respectively. GFP-ILK (Fig. 1 L) and GFP–PINCH-1 (Fig. 1 N) localized to cell–matrix adhesions in both the CH-ILKBP siRNA transfectants (Fig. 1, K and M) and the control transfectants (unpublished data). Although depletion of CH-ILKBP prevented neither the formation of cell–matrix adhesions nor the localization of ILK and PINCH-1 to the adhesion sites, it induced a dramatic increase of apoptosis as revealed by the appearance of a large percentage of TUNEL-positive cells (Fig. 2, A and B). Analyses of the activity of caspase-3, a key mediator of apoptosis, showed that its activity was markedly increased in the absence of CH-ILKBP (Fig. 2 C). These results suggest that (a) CH-ILKBP is essential for cell survival albeit it is not absolutely required for the formation of cell–matrix adhesions and (b) CH-ILKBP acts in the cell survival pathway upstream of caspase-3.

Bottom Line: Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2.However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation.Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

ABSTRACT
Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

Show MeSH
Related in: MedlinePlus