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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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PKB/Akt is the primary target of CH-ILKBP in cell survival. (A and B) CH-ILKBP modulates ERK phosphorylation. The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control transfectants (lanes 2 and 5), and HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and then stimulated with 10% FCS (A, lanes 4–6) or 2 ng/ml IGF-1 (B, lanes 4–6) for 10 min. The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 (p44/42), phosphor-Erk1/2(Thr202/Tyr204) (phosphor-p44/42), and phosphor-p38 MAPK (Thr180/Tyr182), respectively. (C) The myristoylated PKB/Akt-expressing cells (see Fig. 4 F) were transfected with the CH-ILKBP siRNA (lanes 1 and 2) or the control RNA (lanes 3 and 4). The cells were serum starved (lanes 1 and 3) and then stimulated with 2 ng/ml IGF-1 (lanes 2 and 4) for 10 min. The cell lysates (10 μg protein/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 and phosphor-Erk1/2(Thr202/Tyr204), respectively. (D–F) Expression of membrane-bound PKB/Akt bypasses the requirement of CH-ILKBP for cell survival. Apoptotic cells were detected using TUNEL (D), and the percentages of the TUNEL-positive cells (E) were calculated as in the legend to Fig. 2. Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the control RNA; CH-ILKBP(−) + Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the CH-ILKBP siRNA; CH-ILKBP(−) + myr-Akt, HeLa cells transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt and the CH-ILKBP siRNA. (F) The lysates (10 μg/lane) from the transfectants were analyzed by Western blotting with anti–CH-ILKBP mAb 3B5.
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fig5: PKB/Akt is the primary target of CH-ILKBP in cell survival. (A and B) CH-ILKBP modulates ERK phosphorylation. The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control transfectants (lanes 2 and 5), and HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and then stimulated with 10% FCS (A, lanes 4–6) or 2 ng/ml IGF-1 (B, lanes 4–6) for 10 min. The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 (p44/42), phosphor-Erk1/2(Thr202/Tyr204) (phosphor-p44/42), and phosphor-p38 MAPK (Thr180/Tyr182), respectively. (C) The myristoylated PKB/Akt-expressing cells (see Fig. 4 F) were transfected with the CH-ILKBP siRNA (lanes 1 and 2) or the control RNA (lanes 3 and 4). The cells were serum starved (lanes 1 and 3) and then stimulated with 2 ng/ml IGF-1 (lanes 2 and 4) for 10 min. The cell lysates (10 μg protein/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 and phosphor-Erk1/2(Thr202/Tyr204), respectively. (D–F) Expression of membrane-bound PKB/Akt bypasses the requirement of CH-ILKBP for cell survival. Apoptotic cells were detected using TUNEL (D), and the percentages of the TUNEL-positive cells (E) were calculated as in the legend to Fig. 2. Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the control RNA; CH-ILKBP(−) + Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the CH-ILKBP siRNA; CH-ILKBP(−) + myr-Akt, HeLa cells transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt and the CH-ILKBP siRNA. (F) The lysates (10 μg/lane) from the transfectants were analyzed by Western blotting with anti–CH-ILKBP mAb 3B5.

Mentions: Stimulation of cells with serum induced phosphorylation of not only PKB/Akt but also ERK1/2 and p38 MAPK (Fig. 5 A, lanes 5 and 6). Loss of CH-ILKBP modestly inhibited serum induced phosphorylation of ERK1/2 (Fig. 5 A, lane 4 compared with 5 and 6) but not that of p38 MAPK (Fig. 5 A, lanes 4–6). A modest inhibitory effect on IGF-1–induced ERK1/2 phosphorylation has also been observed (Fig. 5 B, lane 4). To test whether PKB/Akt is the primary target of CH-ILKBP in cell survival, we analyzed the effect of expression of the constitutively active PKB/Akt on apoptosis induced by the loss of CH-ILKBP. Expression of myristoylated PKB/Akt, which overrode the inhibition on the membrane translocation, and consequently the activating phosphorylation of PKB/Akt induced by the loss of CH-ILKBP (Fig. 4 F), did not release the modest inhibition of ERK1/2 phosphorylation (Fig. 5 C, lane 2 compared with 4). However, it did rescue cells from apoptosis induced by the loss of CH-ILKBP (Fig. 5, D and E). In control experiments, expression of the constitutively active PKB/Akt did not release the siRNA-mediated suppression of CH-ILKBP expression (Fig. 5 F). Thus, PKB/Akt, and specifically the membrane translocation of PKB/Akt, is the primary target of CH-ILKBP in protection of cells from apoptosis.


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)

PKB/Akt is the primary target of CH-ILKBP in cell survival. (A and B) CH-ILKBP modulates ERK phosphorylation. The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control transfectants (lanes 2 and 5), and HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and then stimulated with 10% FCS (A, lanes 4–6) or 2 ng/ml IGF-1 (B, lanes 4–6) for 10 min. The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 (p44/42), phosphor-Erk1/2(Thr202/Tyr204) (phosphor-p44/42), and phosphor-p38 MAPK (Thr180/Tyr182), respectively. (C) The myristoylated PKB/Akt-expressing cells (see Fig. 4 F) were transfected with the CH-ILKBP siRNA (lanes 1 and 2) or the control RNA (lanes 3 and 4). The cells were serum starved (lanes 1 and 3) and then stimulated with 2 ng/ml IGF-1 (lanes 2 and 4) for 10 min. The cell lysates (10 μg protein/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 and phosphor-Erk1/2(Thr202/Tyr204), respectively. (D–F) Expression of membrane-bound PKB/Akt bypasses the requirement of CH-ILKBP for cell survival. Apoptotic cells were detected using TUNEL (D), and the percentages of the TUNEL-positive cells (E) were calculated as in the legend to Fig. 2. Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the control RNA; CH-ILKBP(−) + Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the CH-ILKBP siRNA; CH-ILKBP(−) + myr-Akt, HeLa cells transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt and the CH-ILKBP siRNA. (F) The lysates (10 μg/lane) from the transfectants were analyzed by Western blotting with anti–CH-ILKBP mAb 3B5.
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fig5: PKB/Akt is the primary target of CH-ILKBP in cell survival. (A and B) CH-ILKBP modulates ERK phosphorylation. The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control transfectants (lanes 2 and 5), and HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and then stimulated with 10% FCS (A, lanes 4–6) or 2 ng/ml IGF-1 (B, lanes 4–6) for 10 min. The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 (p44/42), phosphor-Erk1/2(Thr202/Tyr204) (phosphor-p44/42), and phosphor-p38 MAPK (Thr180/Tyr182), respectively. (C) The myristoylated PKB/Akt-expressing cells (see Fig. 4 F) were transfected with the CH-ILKBP siRNA (lanes 1 and 2) or the control RNA (lanes 3 and 4). The cells were serum starved (lanes 1 and 3) and then stimulated with 2 ng/ml IGF-1 (lanes 2 and 4) for 10 min. The cell lysates (10 μg protein/lane) were analyzed by Western blotting with antibodies recognizing Erk1/2 and phosphor-Erk1/2(Thr202/Tyr204), respectively. (D–F) Expression of membrane-bound PKB/Akt bypasses the requirement of CH-ILKBP for cell survival. Apoptotic cells were detected using TUNEL (D), and the percentages of the TUNEL-positive cells (E) were calculated as in the legend to Fig. 2. Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the control RNA; CH-ILKBP(−) + Vector, HeLa cells transfected with a pUSEamp(+) vector lacking Akt sequence and the CH-ILKBP siRNA; CH-ILKBP(−) + myr-Akt, HeLa cells transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt and the CH-ILKBP siRNA. (F) The lysates (10 μg/lane) from the transfectants were analyzed by Western blotting with anti–CH-ILKBP mAb 3B5.
Mentions: Stimulation of cells with serum induced phosphorylation of not only PKB/Akt but also ERK1/2 and p38 MAPK (Fig. 5 A, lanes 5 and 6). Loss of CH-ILKBP modestly inhibited serum induced phosphorylation of ERK1/2 (Fig. 5 A, lane 4 compared with 5 and 6) but not that of p38 MAPK (Fig. 5 A, lanes 4–6). A modest inhibitory effect on IGF-1–induced ERK1/2 phosphorylation has also been observed (Fig. 5 B, lane 4). To test whether PKB/Akt is the primary target of CH-ILKBP in cell survival, we analyzed the effect of expression of the constitutively active PKB/Akt on apoptosis induced by the loss of CH-ILKBP. Expression of myristoylated PKB/Akt, which overrode the inhibition on the membrane translocation, and consequently the activating phosphorylation of PKB/Akt induced by the loss of CH-ILKBP (Fig. 4 F), did not release the modest inhibition of ERK1/2 phosphorylation (Fig. 5 C, lane 2 compared with 4). However, it did rescue cells from apoptosis induced by the loss of CH-ILKBP (Fig. 5, D and E). In control experiments, expression of the constitutively active PKB/Akt did not release the siRNA-mediated suppression of CH-ILKBP expression (Fig. 5 F). Thus, PKB/Akt, and specifically the membrane translocation of PKB/Akt, is the primary target of CH-ILKBP in protection of cells from apoptosis.

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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Related in: MedlinePlus