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EGF-induced sodium influx regulates EGFR trafficking through HDAC6 and tubulin acetylation.

Lee SJ, Li Z, Litan A, Yoo S, Langhans SA - BMC Cell Biol. (2015)

Bottom Line: Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin.In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover.Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation.

View Article: PubMed Central - PubMed

Affiliation: Nemours Center for Childhood Cancer Research, Alfred I. duPont Hospital for Children, 1701 Rockland Road, Wilmington, DE, 19803, USA. lee346@mailbox.sc.edu.

ABSTRACT

Background: Endocytosis of activated EGF receptor (EGFR) to specific endocytic compartments is required to terminate EGF signaling. Trafficking of EGFR relies on microtubule tracks that transport the cargo vesicle to their intermediate and final destinations and can be modulated through posttranslational modification of tubulin including acetylation. Na,K-ATPase maintains intracellular sodium homeostasis, functions as a signaling scaffold and interacts with EGFR. Na,K-ATPase also binds to and is regulated by acetylated tubulin but whether there is a functional link between EGFR, Na,K-ATPase and tubulin acetylation is not known.

Results: EGF-induced sodium influx regulates EGFR trafficking through increased microtubule acetylation. Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin. In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover. Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation.

Conclusions: These studies provide a novel regulatory mechanism to attenuate EGFR signaling in which EGF modulates EGFR trafficking through intracellular sodium-mediated HDAC6 inactivation and tubulin acetylation.

No MeSH data available.


Related in: MedlinePlus

Sodium influx induces an accumulation of acetylated tubulin. a DAOY cells were incubated with 50 μM ouabain or ethanol (vehicle) for indicated times. Equal amounts of protein were separated by SDS-PAGE and immunoblotted with antibodies recognizing acetylated lysines (upper panel) or acetylated α-tubulin (lower panel). b Immunoblot for acetylated α-tubulin of DAOY cells treated for three hours at indicated concentrations with the sodium ionophore gramicidin A or the Na,K-ATPase inhibitor ouabain. An immunoblot for total α-tubulin ensured equal loading. c DAOY cells were treated for three hours at indicated concentrations with the potassium ionophore valinomycin or the sodium ionophore monensin. Gramicidin A was included for comparison. Equal amounts of protein were used for immunoblotting using antibodies against acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading. d DAOY cells were treated with ouabain or gramicidin A as indicated in isotonic, sodium containing buffer (High Na+) or in a low-sodium buffer in which sodium was substituted with rubidium (Low Na+). Cells were lysed one or three hours after treatment and equal amounts of protein were immunoblotted for total and acetylated α-tubulin. e DAOY cells were incubated for three hours with 50 μM ouabain or vehicle in High Na+ or in Low Na+ buffer. The cells were fixed and immunostained with acetylated α-tubulin and pan-α-tubulin antibodies. For easier comparison, parameters for image acquisition were kept constant between samples. Scale bar, 10 μm. f DAOY cells were pre-incubated with the intracellular calcium chelator BAPTA-AM and then ouabain or gramicidin A were added for three hours. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.
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Fig1: Sodium influx induces an accumulation of acetylated tubulin. a DAOY cells were incubated with 50 μM ouabain or ethanol (vehicle) for indicated times. Equal amounts of protein were separated by SDS-PAGE and immunoblotted with antibodies recognizing acetylated lysines (upper panel) or acetylated α-tubulin (lower panel). b Immunoblot for acetylated α-tubulin of DAOY cells treated for three hours at indicated concentrations with the sodium ionophore gramicidin A or the Na,K-ATPase inhibitor ouabain. An immunoblot for total α-tubulin ensured equal loading. c DAOY cells were treated for three hours at indicated concentrations with the potassium ionophore valinomycin or the sodium ionophore monensin. Gramicidin A was included for comparison. Equal amounts of protein were used for immunoblotting using antibodies against acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading. d DAOY cells were treated with ouabain or gramicidin A as indicated in isotonic, sodium containing buffer (High Na+) or in a low-sodium buffer in which sodium was substituted with rubidium (Low Na+). Cells were lysed one or three hours after treatment and equal amounts of protein were immunoblotted for total and acetylated α-tubulin. e DAOY cells were incubated for three hours with 50 μM ouabain or vehicle in High Na+ or in Low Na+ buffer. The cells were fixed and immunostained with acetylated α-tubulin and pan-α-tubulin antibodies. For easier comparison, parameters for image acquisition were kept constant between samples. Scale bar, 10 μm. f DAOY cells were pre-incubated with the intracellular calcium chelator BAPTA-AM and then ouabain or gramicidin A were added for three hours. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.

Mentions: Although it is now well-documented that acetylated tubulin associates with Na,K-ATPase to inhibit its pump function [18], less is known about the effects of reduced Na,K-ATPase function on tubulin acetylation. To test whether specific inhibition of Na,K-ATPase pump activity modulates the acetylation of tubulin, human DAOY medulloblastoma cells were treated with the cardiac glycoside ouabain. While ouabain specifically binds to Na,K-ATPase and inhibits its function, the ouabain concentrations and duration of treatment used in these experiments does not affect cell viability of DAOY cells [14]. First, to examine the acetylation pattern of whole cell lysates, immunoblotting was performed with an anti-acetylated lysine antibody that specifically recognizes acetylated lysine in a wide range of sequence contexts including acetylated histones, p53 and tubulin. We found little differences in the overall acetylation pattern between control and ouabain-treated cells, except for a noticeable band around 54 kDa that was increased in ouabain-treated cells (Fig. 1a, upper panel). From the size and abundance, we predicted that this band corresponded to acetylated tubulin. Further analyses by immunblotting and immunofluorescence with an anti-acetyl-α-tubulin antibody confirmed the presence of acetylated tubulin in ouabain-treated cells (Fig. 1a, lower panel, and Fig. 1e, upper panels). Since ouabain blocks the Na,K-ATPase activity leading to an increased intracellular sodium level, we next tested the effect of the mechanically distinct sodium ionophores, gramicidin A and monensin, on tubulin acetylation. Gramicidin A is a polypeptide obtained from Bacillus brevis that forms a β − helix inside the hydrophobic interior of the lipid bilayer thereby increasing the permeability of the cell membrane to monovalent cations such as sodium ions [19]. Monensin is a polyether antibiotic that forms a complex with sodium ions to permit these ions to travel across the lipid bilayer [20]. Both gramicidin A (Fig. 1b) and monensin (Fig. 1c) increased the acetylation of tubulin. In contrast, treatment with the potassium ionophore valinomycin did not change the level of acetylated tubulin (Fig. 1c). When NaCl was replaced by KCl the ability of ouabain or gramicidin to induce tubulin acetylation was greatly diminished. (Additional file 1: Figure S1A). Nevertheless, KCl treatment somewhat increased tubulin acetylation but not to the extend observed in ouabain or gramicidin-treated cells. Together these data suggest that an increase in intracellular sodium could result in increased tubulin acetylation. However, we cannot exclude that depolarization of the plasma membrane potential may contribute to the increase in tubulin acetylation observed in ouabain and gramicidin-treated cells.Fig. 1


EGF-induced sodium influx regulates EGFR trafficking through HDAC6 and tubulin acetylation.

Lee SJ, Li Z, Litan A, Yoo S, Langhans SA - BMC Cell Biol. (2015)

Sodium influx induces an accumulation of acetylated tubulin. a DAOY cells were incubated with 50 μM ouabain or ethanol (vehicle) for indicated times. Equal amounts of protein were separated by SDS-PAGE and immunoblotted with antibodies recognizing acetylated lysines (upper panel) or acetylated α-tubulin (lower panel). b Immunoblot for acetylated α-tubulin of DAOY cells treated for three hours at indicated concentrations with the sodium ionophore gramicidin A or the Na,K-ATPase inhibitor ouabain. An immunoblot for total α-tubulin ensured equal loading. c DAOY cells were treated for three hours at indicated concentrations with the potassium ionophore valinomycin or the sodium ionophore monensin. Gramicidin A was included for comparison. Equal amounts of protein were used for immunoblotting using antibodies against acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading. d DAOY cells were treated with ouabain or gramicidin A as indicated in isotonic, sodium containing buffer (High Na+) or in a low-sodium buffer in which sodium was substituted with rubidium (Low Na+). Cells were lysed one or three hours after treatment and equal amounts of protein were immunoblotted for total and acetylated α-tubulin. e DAOY cells were incubated for three hours with 50 μM ouabain or vehicle in High Na+ or in Low Na+ buffer. The cells were fixed and immunostained with acetylated α-tubulin and pan-α-tubulin antibodies. For easier comparison, parameters for image acquisition were kept constant between samples. Scale bar, 10 μm. f DAOY cells were pre-incubated with the intracellular calcium chelator BAPTA-AM and then ouabain or gramicidin A were added for three hours. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4574528&req=5

Fig1: Sodium influx induces an accumulation of acetylated tubulin. a DAOY cells were incubated with 50 μM ouabain or ethanol (vehicle) for indicated times. Equal amounts of protein were separated by SDS-PAGE and immunoblotted with antibodies recognizing acetylated lysines (upper panel) or acetylated α-tubulin (lower panel). b Immunoblot for acetylated α-tubulin of DAOY cells treated for three hours at indicated concentrations with the sodium ionophore gramicidin A or the Na,K-ATPase inhibitor ouabain. An immunoblot for total α-tubulin ensured equal loading. c DAOY cells were treated for three hours at indicated concentrations with the potassium ionophore valinomycin or the sodium ionophore monensin. Gramicidin A was included for comparison. Equal amounts of protein were used for immunoblotting using antibodies against acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading. d DAOY cells were treated with ouabain or gramicidin A as indicated in isotonic, sodium containing buffer (High Na+) or in a low-sodium buffer in which sodium was substituted with rubidium (Low Na+). Cells were lysed one or three hours after treatment and equal amounts of protein were immunoblotted for total and acetylated α-tubulin. e DAOY cells were incubated for three hours with 50 μM ouabain or vehicle in High Na+ or in Low Na+ buffer. The cells were fixed and immunostained with acetylated α-tubulin and pan-α-tubulin antibodies. For easier comparison, parameters for image acquisition were kept constant between samples. Scale bar, 10 μm. f DAOY cells were pre-incubated with the intracellular calcium chelator BAPTA-AM and then ouabain or gramicidin A were added for three hours. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.
Mentions: Although it is now well-documented that acetylated tubulin associates with Na,K-ATPase to inhibit its pump function [18], less is known about the effects of reduced Na,K-ATPase function on tubulin acetylation. To test whether specific inhibition of Na,K-ATPase pump activity modulates the acetylation of tubulin, human DAOY medulloblastoma cells were treated with the cardiac glycoside ouabain. While ouabain specifically binds to Na,K-ATPase and inhibits its function, the ouabain concentrations and duration of treatment used in these experiments does not affect cell viability of DAOY cells [14]. First, to examine the acetylation pattern of whole cell lysates, immunoblotting was performed with an anti-acetylated lysine antibody that specifically recognizes acetylated lysine in a wide range of sequence contexts including acetylated histones, p53 and tubulin. We found little differences in the overall acetylation pattern between control and ouabain-treated cells, except for a noticeable band around 54 kDa that was increased in ouabain-treated cells (Fig. 1a, upper panel). From the size and abundance, we predicted that this band corresponded to acetylated tubulin. Further analyses by immunblotting and immunofluorescence with an anti-acetyl-α-tubulin antibody confirmed the presence of acetylated tubulin in ouabain-treated cells (Fig. 1a, lower panel, and Fig. 1e, upper panels). Since ouabain blocks the Na,K-ATPase activity leading to an increased intracellular sodium level, we next tested the effect of the mechanically distinct sodium ionophores, gramicidin A and monensin, on tubulin acetylation. Gramicidin A is a polypeptide obtained from Bacillus brevis that forms a β − helix inside the hydrophobic interior of the lipid bilayer thereby increasing the permeability of the cell membrane to monovalent cations such as sodium ions [19]. Monensin is a polyether antibiotic that forms a complex with sodium ions to permit these ions to travel across the lipid bilayer [20]. Both gramicidin A (Fig. 1b) and monensin (Fig. 1c) increased the acetylation of tubulin. In contrast, treatment with the potassium ionophore valinomycin did not change the level of acetylated tubulin (Fig. 1c). When NaCl was replaced by KCl the ability of ouabain or gramicidin to induce tubulin acetylation was greatly diminished. (Additional file 1: Figure S1A). Nevertheless, KCl treatment somewhat increased tubulin acetylation but not to the extend observed in ouabain or gramicidin-treated cells. Together these data suggest that an increase in intracellular sodium could result in increased tubulin acetylation. However, we cannot exclude that depolarization of the plasma membrane potential may contribute to the increase in tubulin acetylation observed in ouabain and gramicidin-treated cells.Fig. 1

Bottom Line: Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin.In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover.Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation.

View Article: PubMed Central - PubMed

Affiliation: Nemours Center for Childhood Cancer Research, Alfred I. duPont Hospital for Children, 1701 Rockland Road, Wilmington, DE, 19803, USA. lee346@mailbox.sc.edu.

ABSTRACT

Background: Endocytosis of activated EGF receptor (EGFR) to specific endocytic compartments is required to terminate EGF signaling. Trafficking of EGFR relies on microtubule tracks that transport the cargo vesicle to their intermediate and final destinations and can be modulated through posttranslational modification of tubulin including acetylation. Na,K-ATPase maintains intracellular sodium homeostasis, functions as a signaling scaffold and interacts with EGFR. Na,K-ATPase also binds to and is regulated by acetylated tubulin but whether there is a functional link between EGFR, Na,K-ATPase and tubulin acetylation is not known.

Results: EGF-induced sodium influx regulates EGFR trafficking through increased microtubule acetylation. Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin. In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover. Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation.

Conclusions: These studies provide a novel regulatory mechanism to attenuate EGFR signaling in which EGF modulates EGFR trafficking through intracellular sodium-mediated HDAC6 inactivation and tubulin acetylation.

No MeSH data available.


Related in: MedlinePlus