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

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.

Fig. 1

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.

Fig. 2

An EGF-induced increase in the intracellular sodium level results in increased acetylated tubulin levels. a DAOY cells were incubated with 10 ng/ml EGF or vehicle for indicated time points. Equal amounts of protein were immunoblotted for total and activated (Y1173) EGFR, and acetylated α-tubulin. An immunoblot for total α-tubulin was included to ensure equal loading. b DAOY cells were incubated for one hour in Low Na⁺ buffer and then treated with 10 ng/ml EGF for indicated times. Immunoblots were performed for total and activated (Y1173) EGFR, total and activated Erk1/2, total and activated (S473) Akt and acetylated α-tubulin. The total α-tubulin immunoblot confirmed equal loading. c DAOY cells were incubated for one hour with 10 μM AG1478 prior to addition of 10 ng/ml EGF. Immunoblots were performed as in b. d DAOY cells were incubated in the absence of EGF with either AG1478 or PP2 for one hour before 50 μM ouabain or 100 nM gramicidin A were added for an additional three hours. Equal amounts of protein were immunoblotted for acetylated and total α-tubulin. e DAOY cells were pre-incubated with BAPTA-AM and then 10 ng/ml EGF was added for indicated times. Equal amounts of protein were immunoblotted for acetylated α-tubulin. An immunoblot for total α-tubulin ensured equal loading.

Fig. 3

Intracellular sodium levels regulate vesicle trafficking. a DAOY cells were preincubated with either High Na⁺ or Low Na⁺ buffer for 30 minutes and then stimulated with 40 ng/ml of AlexaFluor 488-conjugated EGF for an additional 15 minutes during which time epifluorescence images were taken with a cooled CCD camera at a rate of one frame/second for a total of 900 frames. The motility of EGF-positive vesicles was analyzed using Slidebook software and average speed, maximum speed and end/total displacement were plotted. *, P < 0.0001. b EGF was added to DAOY cells incubated in buffers of increasing sodium concentrations and the motility of EGF-positive vesicles was analyzed as in A.

Fig. 4

Increased acetylated tubulin decreases the motility of EGF-positive vesicles. a DAOY cells were transiently transfected with either wild-type tubulin (WT) or a nonacetylated mutant of tubulin (K40A). AlexaFluor 488-EGF was added in High Na⁺ buffer and images were acquired as described above. Average speed, maximum speed and the end/total displacement are shown. *, P < 0.001. b DAOY cells were preincubated with 100 nM TSA for 15 minutes and then AlexaFluor 488-EGF was added. Vesicles were tracked as described in figure legend 3. *, P < 0.001.

Fig. 5

Increased intracellular sodium inhibits HDAC6 activity. a DAOY cells were incubated with increasing concentrations of ouabain for three hours and then lysed for an in vitro HDAC activity assay as described in Materials and Methods. b DAOY cells were incubated with indicated concentrations of ouabain and the protein levels of the indicated HDAC isoforms were determined by immunoblotting. Actin immunoblot ensures equal loading. c After incubation with indicated concentrations of gramicidin A, HDAC6 was immunoprecipitated with a specific antibody and then subjected to an in vitro HDAC activity assay using a fluorescent substrate. d DAOY cells were transiently transfected with Flag-HDAC6 cDNA. The cells were incubated with the indicated concentrations of gramicidin A followed by immunoprecipitation of HDAC6. The immunoprecipitates were washed extensively and then incubated with polymerized microtubules to measure deacetylase activity. The reaction was terminated by adding lysis buffer and the samples were processed for immunoblotting with indicated antibodies. e Immunoblot for HDAC6 of stable clones of DAOY cells expressing HDAC6 shRNA. Control cells were transfected with vector only and processed in parallel. A GAPDH blot ensures that equal amounts of protein were used for analysis. f The HDAC6 knockdown cells were incubated with either High Na⁺ or Low Na⁺ buffer and the trafficking patterns of EGF-positive vesicles were analyzed as described in figure legend 3. *, P < 0.0001.

Fig. 6

Acetylated tubulin mediated by intracellular sodium level regulates vesicle trafficking between endosomes. a DAOY cells were treated with AlexaFluor 488-EGF for 30 minutes a, b) or 60 minutes (C, D) in High Na⁺ or Low Na⁺ buffer. Immunofluorescence of early endosomes (EEA1) (A) and late endosomes (LAMP2) c are shown. The squares depict the areas of higher magnification. Scale bars represent 10 μm (low magnification) and 1 μm (high magnification). (B and D) The colocalization ratio was calculated using ImageJ and plotted as mean with standard errors.

Fig. 7

Schematic model. a Resting cell in the absence of ligand. b Ligand-binding leading to increased intracellular sodium and increased tubulin acetylation (mediated through inhibition of HDAC6). c The ligand-receptor complex is internalized and trafficked for degradation. d The ligand-receptor complex is degraded and signaling is terminated. Na,K-ATPase restores low intracellular sodium levels.