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PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways.

Kveiborg M, Instrell R, Rowlands C, Howell M, Parker PJ - PLoS ONE (2011)

Bottom Line: In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF.However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF.Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. marie.kveiborg@bric.ku.dk

ABSTRACT
Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

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PMA-induced ectodomain shedding of HB-EGF from the cell surface.A) Western blot analysis of total cell extracts from HT1080 cells stably expressing alkaline phosphatase tagged HB-EGF (AP-HB-EGF) or mock transfected controls after 30 min treatment with 400 nM PMA or DMSO control. α-Tubulin is used as an internal loading control. B) AP-HB-EGF release measured as alkaline phosphatase activity (absorbance at 405 nm) in conditioned media, and C) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in AP-HB-EGF expressing HT1080 cells after 15, 30, or 60 min treatment with 400 nM PMA (white bars) or DMSO control (black bars). The graphs show average values ± standard error of the mean of triplicate experiments. *p<0.05; **p<0.01, ***p<0.001 after one-way ANOVA with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated, the comparison is relative to the respective control.
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pone-0017168-g001: PMA-induced ectodomain shedding of HB-EGF from the cell surface.A) Western blot analysis of total cell extracts from HT1080 cells stably expressing alkaline phosphatase tagged HB-EGF (AP-HB-EGF) or mock transfected controls after 30 min treatment with 400 nM PMA or DMSO control. α-Tubulin is used as an internal loading control. B) AP-HB-EGF release measured as alkaline phosphatase activity (absorbance at 405 nm) in conditioned media, and C) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in AP-HB-EGF expressing HT1080 cells after 15, 30, or 60 min treatment with 400 nM PMA (white bars) or DMSO control (black bars). The graphs show average values ± standard error of the mean of triplicate experiments. *p<0.05; **p<0.01, ***p<0.001 after one-way ANOVA with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated, the comparison is relative to the respective control.

Mentions: To study the molecular mechanisms of PKC-mediated ADAM protease activation, we used a previously reported cell-based model system for PMA-induced ectodomain shedding of HB-EGF [21]. Specifically, a fusion protein of proHB-EGF with placental alkaline phosphatase inserted in the extracellular domain (AP-HB-EGF) was stably expressed in the HT1080 human fibrosarcoma cell line. Western blot analysis of extracts from untreated cells showed a band at approximately 100 kDa corresponding to the expected size of intact, unmodified (i.e. intracellular) AP-HB-EGF, as well as a less abundant band migrating slightly slower, which appears to be the glycosylated AP-HB-EGF, on the plasma membrane (Figure 1A). PMA-treatment resulted in the disappearance of the glycosylated form of AP-HB-EGF with little or no effect on faster migrating non-glycosylated AP-HB-EGF, indicating that only substrate molecules that had completed transit through the trans-Golgi were cleaved (Figure 1A).


PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways.

Kveiborg M, Instrell R, Rowlands C, Howell M, Parker PJ - PLoS ONE (2011)

PMA-induced ectodomain shedding of HB-EGF from the cell surface.A) Western blot analysis of total cell extracts from HT1080 cells stably expressing alkaline phosphatase tagged HB-EGF (AP-HB-EGF) or mock transfected controls after 30 min treatment with 400 nM PMA or DMSO control. α-Tubulin is used as an internal loading control. B) AP-HB-EGF release measured as alkaline phosphatase activity (absorbance at 405 nm) in conditioned media, and C) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in AP-HB-EGF expressing HT1080 cells after 15, 30, or 60 min treatment with 400 nM PMA (white bars) or DMSO control (black bars). The graphs show average values ± standard error of the mean of triplicate experiments. *p<0.05; **p<0.01, ***p<0.001 after one-way ANOVA with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated, the comparison is relative to the respective control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046143&req=5

pone-0017168-g001: PMA-induced ectodomain shedding of HB-EGF from the cell surface.A) Western blot analysis of total cell extracts from HT1080 cells stably expressing alkaline phosphatase tagged HB-EGF (AP-HB-EGF) or mock transfected controls after 30 min treatment with 400 nM PMA or DMSO control. α-Tubulin is used as an internal loading control. B) AP-HB-EGF release measured as alkaline phosphatase activity (absorbance at 405 nm) in conditioned media, and C) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in AP-HB-EGF expressing HT1080 cells after 15, 30, or 60 min treatment with 400 nM PMA (white bars) or DMSO control (black bars). The graphs show average values ± standard error of the mean of triplicate experiments. *p<0.05; **p<0.01, ***p<0.001 after one-way ANOVA with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated, the comparison is relative to the respective control.
Mentions: To study the molecular mechanisms of PKC-mediated ADAM protease activation, we used a previously reported cell-based model system for PMA-induced ectodomain shedding of HB-EGF [21]. Specifically, a fusion protein of proHB-EGF with placental alkaline phosphatase inserted in the extracellular domain (AP-HB-EGF) was stably expressed in the HT1080 human fibrosarcoma cell line. Western blot analysis of extracts from untreated cells showed a band at approximately 100 kDa corresponding to the expected size of intact, unmodified (i.e. intracellular) AP-HB-EGF, as well as a less abundant band migrating slightly slower, which appears to be the glycosylated AP-HB-EGF, on the plasma membrane (Figure 1A). PMA-treatment resulted in the disappearance of the glycosylated form of AP-HB-EGF with little or no effect on faster migrating non-glycosylated AP-HB-EGF, indicating that only substrate molecules that had completed transit through the trans-Golgi were cleaved (Figure 1A).

Bottom Line: In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF.However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF.Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. marie.kveiborg@bric.ku.dk

ABSTRACT
Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

Show MeSH
Related in: MedlinePlus