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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.

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Effects of ATII on localization and fate of EGFR.A, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, location of AT1AR (green) and endogenous Rab11 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 2ng/ml of EGF for 3 hour with or without 1 hour pretreated with 100nM ATII. Protein level of EGFR was determined by Western Blotting. Blots were stripped and reprobed for Na+K+ATPase to normalize for loading. These results are representative of three independent experiments. Pictures are representative of at least three experiments.
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pone-0080721-g001: Effects of ATII on localization and fate of EGFR.A, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, location of AT1AR (green) and endogenous Rab11 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 2ng/ml of EGF for 3 hour with or without 1 hour pretreated with 100nM ATII. Protein level of EGFR was determined by Western Blotting. Blots were stripped and reprobed for Na+K+ATPase to normalize for loading. These results are representative of three independent experiments. Pictures are representative of at least three experiments.

Mentions: In our previous study, we reported that sustained stimulation with serotonin (5-HT) led to co-sequestration of the 5-HT receptor and EGFR to the pericentrion [7]. To determine if this effect extends to other GPCRs, the effects of AT-II on AT1AR and EGFR were examined. Utilizing HEK cells stably expressing AT1AR as a model system, the effects of AT-II on receptor localization were initially determined. As can be seen, AT1AR predominantly localized on the plasma membrane in control cells, but following prolonged AT-II treatment (60 min), the majority of AT1AR had translocated to a perinuclear region where it partly co-localized with Rab11, a marker of the perinuclear recycling compartment (Figure 1A). Next, the effects of AT-II on localization of both AT-II and EGFR were assessed. Again, most AT1AR and EGFR localized on the plasma membrane in control cells that were serum starved. As seen above, AT-II treatment induced translocation of the AT1AR to the pericentrion. Importantly, AT-II induced translocation of both receptors showing significant co-localization in the perinuclear compartment (Figure 1B).


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 localization and fate of EGFR.A, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, location of AT1AR (green) and endogenous Rab11 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 2ng/ml of EGF for 3 hour with or without 1 hour pretreated with 100nM ATII. Protein level of EGFR was determined by Western Blotting. Blots were stripped and reprobed for Na+K+ATPase to normalize for loading. These results are representative of three independent experiments. Pictures are representative of at least three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0080721-g001: Effects of ATII on localization and fate of EGFR.A, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, location of AT1AR (green) and endogenous Rab11 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells stably transfected with AT1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells stably transfected with AT1AR were serum starved for 5 hours and treated with 2ng/ml of EGF for 3 hour with or without 1 hour pretreated with 100nM ATII. Protein level of EGFR was determined by Western Blotting. Blots were stripped and reprobed for Na+K+ATPase to normalize for loading. These results are representative of three independent experiments. Pictures are representative of at least three experiments.
Mentions: In our previous study, we reported that sustained stimulation with serotonin (5-HT) led to co-sequestration of the 5-HT receptor and EGFR to the pericentrion [7]. To determine if this effect extends to other GPCRs, the effects of AT-II on AT1AR and EGFR were examined. Utilizing HEK cells stably expressing AT1AR as a model system, the effects of AT-II on receptor localization were initially determined. As can be seen, AT1AR predominantly localized on the plasma membrane in control cells, but following prolonged AT-II treatment (60 min), the majority of AT1AR had translocated to a perinuclear region where it partly co-localized with Rab11, a marker of the perinuclear recycling compartment (Figure 1A). Next, the effects of AT-II on localization of both AT-II and EGFR were assessed. Again, most AT1AR and EGFR localized on the plasma membrane in control cells that were serum starved. As seen above, AT-II treatment induced translocation of the AT1AR to the pericentrion. Importantly, AT-II induced translocation of both receptors showing significant co-localization in the perinuclear compartment (Figure 1B).

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