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 ATII on phosphorylation of EGFR.A, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours followed by 100nM ATII for 10 min. Phosphorylation of EGFR on Thr-654 (P-Thr654) was determined by western blotting. Blots were stripped and reprobed for total EGFR to normalize for loading. B, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 5ng/ml of EGF for 5 min with or without 1 hour pretreated with 100nM ATII. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) was determined by western blotting. The blots were stripped and reprobed for total EGFR to normalize for loading. C, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, FIPI or vehicle followed with 100nM ATII or vehicle for 1 hour and then treated with 5ng/ml EGF for 5 min. P-Tyr1045 and EGFR were determined by western blotting. D, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, 1-butanol or vehicle followed with 100nM ATII or vehicle for 5min, P-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-g003: Effects of ATII on phosphorylation of EGFR.A, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours followed by 100nM ATII for 10 min. Phosphorylation of EGFR on Thr-654 (P-Thr654) was determined by western blotting. Blots were stripped and reprobed for total EGFR to normalize for loading. B, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 5ng/ml of EGF for 5 min with or without 1 hour pretreated with 100nM ATII. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) was determined by western blotting. The blots were stripped and reprobed for total EGFR to normalize for loading. C, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, FIPI or vehicle followed with 100nM ATII or vehicle for 1 hour and then treated with 5ng/ml EGF for 5 min. P-Tyr1045 and EGFR were determined by western blotting. D, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, 1-butanol or vehicle followed with 100nM ATII or vehicle for 5min, P-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.

Mentions: Previous research has reported that phosphorylation of EGFR at various residues is important for regulating its trafficking. Indeed, PMA-induced phosphorylation of EGFR on Thr-654 was reported to change its fate from the lysosomes to the recycling endosomes [18]. In contrast, phosphorylation of EGFR on tyrosine 1045 (Tyr-1045) was found to be necessary for binding to c-Cbl, receptor ubiquitination, and degradation [23]. As the pericentrion is a subset of recycling endosomes and is required for the AT-II-induced inhibition, it became important to determine the role of these phosphorylation sites in this process. Initially, the effects of AT-II on phosphorylation of EGFR at Thr-654 and Tyr-1045 were determined. As can be seen (Figure 3A), there was a basal phosphorylation of Thr-654 in unstimulated cells that was strongly increased by AT-II treatment. In contrast, basal phosphorylation of EGFR at Tyr-1045 was minimal and was sharply increased by EGF treatment, consistent with induction of EGFR degradation. However, while AT-II had no effect on basal Tyr-1045 phosphorylation, pretreatment of cells with AT-II partially inhibited the phosphorylation of Tyr-1045 induced by EGF (Figure 3B). As Tyr-1045 phosphorylation is important for EGFR degradation, this could account for the observed effect of AT-II on the inhibition of the loss of EGFR. If this were the case and, given results suggesting that EGFR sequestration in the pericentrion is required to prevent EGFR degradation (Figure 2E), we reasoned that the effect of AT-II on Tyr-1045 would be sensitive to inhibitors of pericentrion formation; consequently, cells were treated with inhibitors of cPKC (Gö6976) and PLD (FIPI) (Figure 3C). The results showed that inhibition of either PKC or PLD [24,25] did not prevent the effects of AT-II on Tyr-1045 phosphorylation, thereby suggesting that Tyr-1045 phosphorylation occurs independently 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 ATII on phosphorylation of EGFR.A, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours followed by 100nM ATII for 10 min. Phosphorylation of EGFR on Thr-654 (P-Thr654) was determined by western blotting. Blots were stripped and reprobed for total EGFR to normalize for loading. B, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 5ng/ml of EGF for 5 min with or without 1 hour pretreated with 100nM ATII. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) was determined by western blotting. The blots were stripped and reprobed for total EGFR to normalize for loading. C, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, FIPI or vehicle followed with 100nM ATII or vehicle for 1 hour and then treated with 5ng/ml EGF for 5 min. P-Tyr1045 and EGFR were determined by western blotting. D, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, 1-butanol or vehicle followed with 100nM ATII or vehicle for 5min, P-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-g003: Effects of ATII on phosphorylation of EGFR.A, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours followed by 100nM ATII for 10 min. Phosphorylation of EGFR on Thr-654 (P-Thr654) was determined by western blotting. Blots were stripped and reprobed for total EGFR to normalize for loading. B, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 5ng/ml of EGF for 5 min with or without 1 hour pretreated with 100nM ATII. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) was determined by western blotting. The blots were stripped and reprobed for total EGFR to normalize for loading. C, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, FIPI or vehicle followed with 100nM ATII or vehicle for 1 hour and then treated with 5ng/ml EGF for 5 min. P-Tyr1045 and EGFR were determined by western blotting. D, HEK293 cells stably transfected with AT1AR were serum starved for 5hours and then pretreated with Gö6976, 1-butanol or vehicle followed with 100nM ATII or vehicle for 5min, P-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.
Mentions: Previous research has reported that phosphorylation of EGFR at various residues is important for regulating its trafficking. Indeed, PMA-induced phosphorylation of EGFR on Thr-654 was reported to change its fate from the lysosomes to the recycling endosomes [18]. In contrast, phosphorylation of EGFR on tyrosine 1045 (Tyr-1045) was found to be necessary for binding to c-Cbl, receptor ubiquitination, and degradation [23]. As the pericentrion is a subset of recycling endosomes and is required for the AT-II-induced inhibition, it became important to determine the role of these phosphorylation sites in this process. Initially, the effects of AT-II on phosphorylation of EGFR at Thr-654 and Tyr-1045 were determined. As can be seen (Figure 3A), there was a basal phosphorylation of Thr-654 in unstimulated cells that was strongly increased by AT-II treatment. In contrast, basal phosphorylation of EGFR at Tyr-1045 was minimal and was sharply increased by EGF treatment, consistent with induction of EGFR degradation. However, while AT-II had no effect on basal Tyr-1045 phosphorylation, pretreatment of cells with AT-II partially inhibited the phosphorylation of Tyr-1045 induced by EGF (Figure 3B). As Tyr-1045 phosphorylation is important for EGFR degradation, this could account for the observed effect of AT-II on the inhibition of the loss of EGFR. If this were the case and, given results suggesting that EGFR sequestration in the pericentrion is required to prevent EGFR degradation (Figure 2E), we reasoned that the effect of AT-II on Tyr-1045 would be sensitive to inhibitors of pericentrion formation; consequently, cells were treated with inhibitors of cPKC (Gö6976) and PLD (FIPI) (Figure 3C). The results showed that inhibition of either PKC or PLD [24,25] did not prevent the effects of AT-II on Tyr-1045 phosphorylation, thereby suggesting that Tyr-1045 phosphorylation occurs independently 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