Limits...
Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

Liu M, Idkowiak-Baldys J, Roddy PL, Baldys A, Raymond J, Clarke CJ, Hannun YA - PLoS ONE (2013)

Bottom Line: Thus, Thr-654 phosphorylation required the formation of the pericentrion.On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF.Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and The Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York, United States of America ; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT
It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

Show MeSH

Related in: MedlinePlus

Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3823608&req=5

pone-0080721-g004: Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.

Mentions: Thus far, the results demonstrated a role for the pericentrion in the AT-II induced protection of EGFR, and suggested that this is not through effects on Tyr-1045 phosphorylation. As previous results have reported that phosphorylation of EGFR at Thr-654 is PKC-mediated and is important for regulating EGFR fate [18], it became important to determine the role of the pericentrion in this process. Initially, the effects of PMA on Thr-654 phosphorylation were determined. As can be seen (Figure 4A), PMA initiated phosphorylation of Thr-654 at 10 min with levels being sustained for the course of the experiment (Figure 4A). Importantly, this time course of phosphorylation is significantly later than initial events mediated by PKC at the plasma membrane (which occurs within 30-60 seconds) and is more coincident with formation of the pericentrion.


Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

Liu M, Idkowiak-Baldys J, Roddy PL, Baldys A, Raymond J, Clarke CJ, Hannun YA - PLoS ONE (2013)

Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823608&req=5

pone-0080721-g004: Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.
Mentions: Thus far, the results demonstrated a role for the pericentrion in the AT-II induced protection of EGFR, and suggested that this is not through effects on Tyr-1045 phosphorylation. As previous results have reported that phosphorylation of EGFR at Thr-654 is PKC-mediated and is important for regulating EGFR fate [18], it became important to determine the role of the pericentrion in this process. Initially, the effects of PMA on Thr-654 phosphorylation were determined. As can be seen (Figure 4A), PMA initiated phosphorylation of Thr-654 at 10 min with levels being sustained for the course of the experiment (Figure 4A). Importantly, this time course of phosphorylation is significantly later than initial events mediated by PKC at the plasma membrane (which occurs within 30-60 seconds) and is more coincident with formation of the pericentrion.

Bottom Line: Thus, Thr-654 phosphorylation required the formation of the pericentrion.On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF.Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and The Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York, United States of America ; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT
It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

Show MeSH
Related in: MedlinePlus