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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 regulates PKB/Akt phosphorylation. (A and B) The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control RNA transfectants (lanes 2 and 5), and the parental HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and 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 Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), phosphor-PDK1(Ser241), and PDK1, respectively. (C) The CH-ILKBP siRNA transfectants (lanes 1 and 2), the control RNA transfectants (lanes 3 and 4), and HeLa cells (lanes 5 and 6) were kept in suspension for 2 h and then either harvested (lanes 1, 3, and 5) or allowed to adhere on laminin-coated plates for 30 min (lanes 2, 4, and 6). The cell lysates (15 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), and phospho-Akt(Thr308), respectively.
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fig3: CH-ILKBP regulates PKB/Akt phosphorylation. (A and B) The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control RNA transfectants (lanes 2 and 5), and the parental HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and 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 Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), phosphor-PDK1(Ser241), and PDK1, respectively. (C) The CH-ILKBP siRNA transfectants (lanes 1 and 2), the control RNA transfectants (lanes 3 and 4), and HeLa cells (lanes 5 and 6) were kept in suspension for 2 h and then either harvested (lanes 1, 3, and 5) or allowed to adhere on laminin-coated plates for 30 min (lanes 2, 4, and 6). The cell lysates (15 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), and phospho-Akt(Thr308), respectively.

Mentions: To gain insight into the mechanism by which CH-ILKBP functions in cell survival, we analyzed the effect of loss of CH-ILKBP on the activating phosphorylation of PKB/Akt, a key membrane-proximal survival signaling intermediate that is activated in response to growth factors and cell–matrix adhesion. Stimulation of HeLa cells with serum (Fig. 3 A, lane 6) or insulin-like growth factor (IGF)-1 (Fig. 3 B, lane 6) induced phosphorylation of PKB/Akt at both Ser473 and Thr308. However, the activating phosphorylation of PKB/Akt was impaired in the CH-ILKBP–deficient cells (Fig. 3, A and B, lane 4 compared with 6) but not the control cells (Fig. 3, A and B, lane 5 compared with 6). In parallel experiments, a similar amount of PKB/Akt protein was detected in the CH-ILKBP–deficient cells and the control cells (Fig. 3, A and B, lanes 1–6). Loss of CH-ILKBP did not alter the protein level or phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1) (Fig. 3, A and B), an upstream Ser/Thr kinase responsible for the Thr308 phosphorylation of membrane-bound PKB/Akt. Collectively, these results suggest that CH-ILKBP is critically involved in the cellular phosphorylation of PKB/Akt without altering the protein level or phosphorylation of PDK1.


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 regulates PKB/Akt phosphorylation. (A and B) The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control RNA transfectants (lanes 2 and 5), and the parental HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and 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 Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), phosphor-PDK1(Ser241), and PDK1, respectively. (C) The CH-ILKBP siRNA transfectants (lanes 1 and 2), the control RNA transfectants (lanes 3 and 4), and HeLa cells (lanes 5 and 6) were kept in suspension for 2 h and then either harvested (lanes 1, 3, and 5) or allowed to adhere on laminin-coated plates for 30 min (lanes 2, 4, and 6). The cell lysates (15 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), and phospho-Akt(Thr308), respectively.
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Related In: Results  -  Collection

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fig3: CH-ILKBP regulates PKB/Akt phosphorylation. (A and B) The CH-ILKBP siRNA transfectants (lanes 1 and 4), the control RNA transfectants (lanes 2 and 5), and the parental HeLa cells (lanes 3 and 6) were serum starved (lanes 1–3) and 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 Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), phosphor-PDK1(Ser241), and PDK1, respectively. (C) The CH-ILKBP siRNA transfectants (lanes 1 and 2), the control RNA transfectants (lanes 3 and 4), and HeLa cells (lanes 5 and 6) were kept in suspension for 2 h and then either harvested (lanes 1, 3, and 5) or allowed to adhere on laminin-coated plates for 30 min (lanes 2, 4, and 6). The cell lysates (15 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), and phospho-Akt(Thr308), respectively.
Mentions: To gain insight into the mechanism by which CH-ILKBP functions in cell survival, we analyzed the effect of loss of CH-ILKBP on the activating phosphorylation of PKB/Akt, a key membrane-proximal survival signaling intermediate that is activated in response to growth factors and cell–matrix adhesion. Stimulation of HeLa cells with serum (Fig. 3 A, lane 6) or insulin-like growth factor (IGF)-1 (Fig. 3 B, lane 6) induced phosphorylation of PKB/Akt at both Ser473 and Thr308. However, the activating phosphorylation of PKB/Akt was impaired in the CH-ILKBP–deficient cells (Fig. 3, A and B, lane 4 compared with 6) but not the control cells (Fig. 3, A and B, lane 5 compared with 6). In parallel experiments, a similar amount of PKB/Akt protein was detected in the CH-ILKBP–deficient cells and the control cells (Fig. 3, A and B, lanes 1–6). Loss of CH-ILKBP did not alter the protein level or phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1) (Fig. 3, A and B), an upstream Ser/Thr kinase responsible for the Thr308 phosphorylation of membrane-bound PKB/Akt. Collectively, these results suggest that CH-ILKBP is critically involved in the cellular phosphorylation of PKB/Akt without altering the protein level or phosphorylation of PDK1.

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