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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 PMA on EGFR T654A mutant.A, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation, cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then pretreated with 100nM PMA or vehicle for 1 hour followed by 5ng/ml EGF for 3 hours. The levels of EGFR, EGFR-GFP and actin were determined by western blotting. For all figures, * p <0.05, from at least three independent experiments.
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pone-0080721-g005: Effects of PMA on EGFR T654A mutant.A, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation, cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then pretreated with 100nM PMA or vehicle for 1 hour followed by 5ng/ml EGF for 3 hours. The levels of EGFR, EGFR-GFP and actin were determined by western blotting. For all figures, * p <0.05, from at least three independent experiments.

Mentions: The above results suggest that sequestration, Thr-654 phosphorylation, and protection of EGFR induced by AT-II are all pericentrion-dependent. Therefore, it became important to determine the mechanistic interactions of these effects and, more specifically, if phosphorylation of Thr-654 is essential for either protection or sequestration, and if so, then what is the mechanistic order of these events. To investigate this, a non-phosphorylatable EGFR-T654A mutant (TA-EGFR-GFP) was generated, and the effects of PMA on trafficking of this mutant were compared to wild-type EGFR (WT-EGFR-GP). As seen above, PMA induced translocation of WT-EGFR-GFP to the perinuclear region; strikingly, this was also observed for the mutant TA-EGFR-GFP to the perinuclear region (Figure 5A), demonstrating that Thr-654 phosphorylation is not required for sequestration, and also consistent with the results suggesting that sequestration of EGFR to the pericentrion is necessary for phosphorylation. Importantly, EGF was able to induce loss of TA-EGFR as with WT-EGFR, and with pretreatment of PMA EGF was able to induce the loss of TA-EGFR mutant (Figure 5B). This confirms that Thr-654 phosphorylation is not necessary for its translocation to the pericentrion but is essential for the protection. Thus, the formation of the pericentrion is required for phosphorylation on T654, which in turn is required for the protection of EGFR from accessed by EGF (Figure 6).


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 EGFR T654A mutant.A, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation, cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then pretreated with 100nM PMA or vehicle for 1 hour followed by 5ng/ml EGF for 3 hours. The levels of EGFR, EGFR-GFP and actin were determined by western blotting. For all figures, * p <0.05, from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0080721-g005: Effects of PMA on EGFR T654A mutant.A, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation, cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then pretreated with 100nM PMA or vehicle for 1 hour followed by 5ng/ml EGF for 3 hours. The levels of EGFR, EGFR-GFP and actin were determined by western blotting. For all figures, * p <0.05, from at least three independent experiments.
Mentions: The above results suggest that sequestration, Thr-654 phosphorylation, and protection of EGFR induced by AT-II are all pericentrion-dependent. Therefore, it became important to determine the mechanistic interactions of these effects and, more specifically, if phosphorylation of Thr-654 is essential for either protection or sequestration, and if so, then what is the mechanistic order of these events. To investigate this, a non-phosphorylatable EGFR-T654A mutant (TA-EGFR-GFP) was generated, and the effects of PMA on trafficking of this mutant were compared to wild-type EGFR (WT-EGFR-GP). As seen above, PMA induced translocation of WT-EGFR-GFP to the perinuclear region; strikingly, this was also observed for the mutant TA-EGFR-GFP to the perinuclear region (Figure 5A), demonstrating that Thr-654 phosphorylation is not required for sequestration, and also consistent with the results suggesting that sequestration of EGFR to the pericentrion is necessary for phosphorylation. Importantly, EGF was able to induce loss of TA-EGFR as with WT-EGFR, and with pretreatment of PMA EGF was able to induce the loss of TA-EGFR mutant (Figure 5B). This confirms that Thr-654 phosphorylation is not necessary for its translocation to the pericentrion but is essential for the protection. Thus, the formation of the pericentrion is required for phosphorylation on T654, which in turn is required for the protection of EGFR from accessed by EGF (Figure 6).

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