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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

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.
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Fig4: 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.

Mentions: To test whether the sodium-dependent change in the level of acetylated tubulin mediates the increase in the speed of EGF-positive vesicles, we first inhibited tubulin acetylation using a dominant negative mutant of α-tubulin in which the predominately acetylated lysine is mutated to alanine (K40A) [22]. The K40A tubulin mutant was transiently expressed in DAOY cells and the trafficking pattern of the EGF-positive vesicles was determined. As compared to control cells, expression of the K40A tubulin mutant resulted in a statistically significant increase in the average and the maximum speed of EGF-positive vesicles but did not significantly affect the end/total displacement (Fig. 4a). On the contrary, increasing the level of acetylated tubulin by inhibition of HDAC6 with the pan-HDAC inhibitor trichostatin A (TSA) almost completely abolished the increase in vesicle speed observed in Low Na+ and the levels were similar to that of control cells in High Na+ (Fig. 4b). TSA had a minimal effect on the speed of EGF-positive vesicles in High Na+ and, as expected, did not significantly affect end/total displacement in both Low Na+ and High Na+ (Fig. 4b). Taken together, these data strongly support our hypothesis that trafficking of EGF-containing vesicles is precisely regulated by altering the acetylation status of microtubules through regulating HDAC activity in a sodium-dependent manner.Fig. 4


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)

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.
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Fig4: 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.
Mentions: To test whether the sodium-dependent change in the level of acetylated tubulin mediates the increase in the speed of EGF-positive vesicles, we first inhibited tubulin acetylation using a dominant negative mutant of α-tubulin in which the predominately acetylated lysine is mutated to alanine (K40A) [22]. The K40A tubulin mutant was transiently expressed in DAOY cells and the trafficking pattern of the EGF-positive vesicles was determined. As compared to control cells, expression of the K40A tubulin mutant resulted in a statistically significant increase in the average and the maximum speed of EGF-positive vesicles but did not significantly affect the end/total displacement (Fig. 4a). On the contrary, increasing the level of acetylated tubulin by inhibition of HDAC6 with the pan-HDAC inhibitor trichostatin A (TSA) almost completely abolished the increase in vesicle speed observed in Low Na+ and the levels were similar to that of control cells in High Na+ (Fig. 4b). TSA had a minimal effect on the speed of EGF-positive vesicles in High Na+ and, as expected, did not significantly affect end/total displacement in both Low Na+ and High Na+ (Fig. 4b). Taken together, these data strongly support our hypothesis that trafficking of EGF-containing vesicles is precisely regulated by altering the acetylation status of microtubules through regulating HDAC activity in a sodium-dependent manner.Fig. 4

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