Limits...
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.

Show MeSH

Related in: MedlinePlus

Loss of CH-ILKBP impairs the membrane translocation of PKB/Akt. (A–E) The CH-ILKBP siRNA-transfectants (lanes 1 and 2), the control transfectants (lanes 3 and 4), and the parental HeLa cells (lanes 5 and 6) were serum starved (lanes 1, 3, and 5) or stimulated with 2 ng/ml IGF-1 for 5 min (lanes 2, 4, and 6). Samples from the cytosol (10 μg proteins/lane) and the membrane (corresponding to 20 μg cytosol proteins/lane) fractions were analyzed by Western blotting with antibodies recognizing Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), CH-ILKBP (D), and PDK1 (E), respectively. (F) HeLa cells were transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt (lanes 1–4) or a pUSEamp(+) vector lacking Akt sequence as a control (lanes 5–8). The myristoylated PKB/Akt and the control vector transfectants were then transfected with the CH-ILKBP siRNA (lanes 1, 2, 5, and 6) or the control RNA (lanes 3, 4, 7, and 8). The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), and CH-ILKBP, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172761&req=5

fig4: Loss of CH-ILKBP impairs the membrane translocation of PKB/Akt. (A–E) The CH-ILKBP siRNA-transfectants (lanes 1 and 2), the control transfectants (lanes 3 and 4), and the parental HeLa cells (lanes 5 and 6) were serum starved (lanes 1, 3, and 5) or stimulated with 2 ng/ml IGF-1 for 5 min (lanes 2, 4, and 6). Samples from the cytosol (10 μg proteins/lane) and the membrane (corresponding to 20 μg cytosol proteins/lane) fractions were analyzed by Western blotting with antibodies recognizing Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), CH-ILKBP (D), and PDK1 (E), respectively. (F) HeLa cells were transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt (lanes 1–4) or a pUSEamp(+) vector lacking Akt sequence as a control (lanes 5–8). The myristoylated PKB/Akt and the control vector transfectants were then transfected with the CH-ILKBP siRNA (lanes 1, 2, 5, and 6) or the control RNA (lanes 3, 4, 7, and 8). The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), and CH-ILKBP, respectively.

Mentions: We next investigated the mechanism by which CH-ILKBP functions in the activating phosphorylation of PKB/Akt. Because membrane translocation of PKB/Akt precedes and is essential for the activating phosphorylation of PKB/Akt (Andjelkovic et al., 1997; Scheid et al., 2002), we tested whether CH-ILKBP is required for the membrane translocation of PKB/Akt. Stimulation of HeLa cells or the control cells with IGF-1 induced translocation of PKB/Akt from cytosol to the membrane (Fig. 4 A, lanes 4 and 6), where it was phosphorylated at Ser473 (Fig. 4 B, lanes 4 and 6) and Thr308 (Fig. 4 C, lanes 4 and 6). By marked contrast, the PKB/Akt membrane translocation (Fig. 4 A, lane 2) and phosphorylation at Ser473 (Fig. 4 B, lane 2) and Thr308 (Fig. 4 C, lane 2) were impaired in the absence of CH-ILKBP. Probing the samples with an anti–CH-ILKBP antibody confirmed that CH-ILKBP was depleted in the membrane and the cytosol fractions of the CH-ILKBP siRNA transfectants (Fig. 4 D, lanes 1 and 2). Loss of CH-ILKBP did not significantly alter the amount of PDK1 associated with the membrane (Fig. 4 E, lanes 1 and 2). These results indicate that CH-ILKBP is required for the membrane translocation of PKB/Akt but not the association of PDK1 with the membrane.


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)

Loss of CH-ILKBP impairs the membrane translocation of PKB/Akt. (A–E) The CH-ILKBP siRNA-transfectants (lanes 1 and 2), the control transfectants (lanes 3 and 4), and the parental HeLa cells (lanes 5 and 6) were serum starved (lanes 1, 3, and 5) or stimulated with 2 ng/ml IGF-1 for 5 min (lanes 2, 4, and 6). Samples from the cytosol (10 μg proteins/lane) and the membrane (corresponding to 20 μg cytosol proteins/lane) fractions were analyzed by Western blotting with antibodies recognizing Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), CH-ILKBP (D), and PDK1 (E), respectively. (F) HeLa cells were transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt (lanes 1–4) or a pUSEamp(+) vector lacking Akt sequence as a control (lanes 5–8). The myristoylated PKB/Akt and the control vector transfectants were then transfected with the CH-ILKBP siRNA (lanes 1, 2, 5, and 6) or the control RNA (lanes 3, 4, 7, and 8). The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), and CH-ILKBP, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172761&req=5

fig4: Loss of CH-ILKBP impairs the membrane translocation of PKB/Akt. (A–E) The CH-ILKBP siRNA-transfectants (lanes 1 and 2), the control transfectants (lanes 3 and 4), and the parental HeLa cells (lanes 5 and 6) were serum starved (lanes 1, 3, and 5) or stimulated with 2 ng/ml IGF-1 for 5 min (lanes 2, 4, and 6). Samples from the cytosol (10 μg proteins/lane) and the membrane (corresponding to 20 μg cytosol proteins/lane) fractions were analyzed by Western blotting with antibodies recognizing Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), CH-ILKBP (D), and PDK1 (E), respectively. (F) HeLa cells were transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt (lanes 1–4) or a pUSEamp(+) vector lacking Akt sequence as a control (lanes 5–8). The myristoylated PKB/Akt and the control vector transfectants were then transfected with the CH-ILKBP siRNA (lanes 1, 2, 5, and 6) or the control RNA (lanes 3, 4, 7, and 8). The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), and CH-ILKBP, respectively.
Mentions: We next investigated the mechanism by which CH-ILKBP functions in the activating phosphorylation of PKB/Akt. Because membrane translocation of PKB/Akt precedes and is essential for the activating phosphorylation of PKB/Akt (Andjelkovic et al., 1997; Scheid et al., 2002), we tested whether CH-ILKBP is required for the membrane translocation of PKB/Akt. Stimulation of HeLa cells or the control cells with IGF-1 induced translocation of PKB/Akt from cytosol to the membrane (Fig. 4 A, lanes 4 and 6), where it was phosphorylated at Ser473 (Fig. 4 B, lanes 4 and 6) and Thr308 (Fig. 4 C, lanes 4 and 6). By marked contrast, the PKB/Akt membrane translocation (Fig. 4 A, lane 2) and phosphorylation at Ser473 (Fig. 4 B, lane 2) and Thr308 (Fig. 4 C, lane 2) were impaired in the absence of CH-ILKBP. Probing the samples with an anti–CH-ILKBP antibody confirmed that CH-ILKBP was depleted in the membrane and the cytosol fractions of the CH-ILKBP siRNA transfectants (Fig. 4 D, lanes 1 and 2). Loss of CH-ILKBP did not significantly alter the amount of PDK1 associated with the membrane (Fig. 4 E, lanes 1 and 2). These results indicate that CH-ILKBP is required for the membrane translocation of PKB/Akt but not the association of PDK1 with the membrane.

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