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. 2015 Dec 18;47(12):e200.
doi: 10.1038/emm.2015.101.

The Pleckstrin Homology Domain of Phospholipase D1 Accelerates EGFR Endocytosis by Increasing the Expression of the Rab5 Effector, rabaptin-5

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Free PMC article

The Pleckstrin Homology Domain of Phospholipase D1 Accelerates EGFR Endocytosis by Increasing the Expression of the Rab5 Effector, rabaptin-5

Mi Hee Park et al. Exp Mol Med. .
Free PMC article

Abstract

Endocytosis is differentially regulated by hypoxia-inducible factor-1α (HIF-1α) and phospholipase D (PLD). However, the relationship between HIF-1α and PLD in endocytosis is unknown. HIF-1α is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1α independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1α, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1α, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1α in the EGFR endocytosis pathway.

Figures

Figure 1
Figure 1
PLD1 recovers HIF-1α-mediated inhibition of EGFR endocytosis via the Rab5/7 pathway. (a) HEK293 cells were transfected with GFP–HIF-1α and PLD1, then incubated in serum-free media for 14 h and stimulated with EGF (100 ng ml−1) for 1 h. The indicated proteins were analyzed by western blot. The values were normalized to that of α-tubulin and expressed relative to the control. (b) After HEK293 cells were transfected with GFP–HIF-1α and/or PLD1, they were treated with Alexa Fluor 555–EGF (20 ng ml−1) for the indicated times, fixed and examined by fluorescence microscopy. (c) HEK293 cells were transfected with GFP–Rab5 (Q79L), wtPLD1, mtPLD1 and HA–HIF-1α, then visualized by fluorescence microscopy. Data are representative of three independent experiments. EGFR, epidermal growth factor receptor; HA, hemagglutanin; HIF-1α, hypoxia-inducible factor-1α PLD, phospholipase D.
Figure 2
Figure 2
PLD1 regulates the expression of the Rab5 effector, rabaptin-5, through HIF-1α in an oxygen-dependent manner. (a) Quantitative RT–PCR of rabaptin-5 mRNA in HEK293 cells transfected with the indicated constructs. *P<0.05. (b) Quantitative RT–PCR of rabaptin-5 mRNA in HEK293 cells transfected with PLD1 or siRNA for HIF-1α. *P<0.05. (c) ChIP assay of the binding of HIF-1α to the RABEP1 promoter in HEK293 cells transfected with PLD1. (d) Quantitative RT–PCR assay of rabaptin-5 mRNA in HEK293 cells transfected with the indicated constructs under hypoxia or normoxia. Results are shown as the mean±s.d. of the three independent experiments. EGFR, epidermal growth factor receptor; HIF-1α, hypoxia-inducible factor-1α ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; NS, non-significant; PLD, phospholipase D; RT–PCR, reverse transcription–PCR.
Figure 3
Figure 3
PLD1 promotes EGFR endocytosis by destabilizing HIF-1α protein via the PHD2/VHL-mediated pathway. (a) HEK293 cells were transfected with mtPLD1, then incubated under hypoxia. After hypoxia for 4 h, the cells were reoxygenized and treated with EGF (100 ng ml−1) for 30 min. The lysates were then analyzed by immunoblot. The values were normalized to that of α-tubulin and expressed as a fold of the control. (b and c) HEK293 cells cotransfected with the indicated constructs and stimulated with EGF (100 ng ml−1) for 30 min in the presence MG132. The lysates were immunoprecipitated with anti-PLD (b) or anti-GFP (c) and immunoblotted with the indicated antibodies. The values were normalized against that of α-tubulin and expressed as a fold of the control. (d) Immunoblot assay of the indicated proteins in HEK293 cells transfected with mt PLD1 and stimulated with EGF in the presence MG132. The hydroxylated values were normalized to total HIF-1α and expressed as a fold of the control. (e) HEK293 cells transfected with mt PLD1 and stimulated with EGF in the presence MG132. The lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Data are representative of three independent experiments. EGFR, epidermal growth factor receptor; HIF-1α, hypoxia-inducible factor-1α GFP, green fluorescent protein; PLD, phospholipase D; VHL, von Hippel–Lindau.
Figure 4
Figure 4
PH-domain of PLD1 is crucial for EGFR endocytosis by increasing the level of rabaptin-5. (a) HEK293 cells transfected with GFP–PLD1-PH and incubated under hypoxia. After hypoxia for 4 h, the cells were reoxygenized with treatment of EGF (100 ng ml−1) for 30 min. The values were normalized to that of α-tubulin and expressed as a fold of the control. (b) HEK293 cells were cotransfected with the indicated constructs, then exposed to EGF (100 ng ml−1) for 30 min in the presence of MG132. The lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (c) Immunoblot assay of EGFR under the indicated condition. The values were normalized to that of α-tubulin and expressed as a fold of the control. (d) HEK293 cells were cotransfected with GFP–HIF-1α and GFP–PLD1-PH. After 24 h of serum starvation, the cells were treated with Alexa Fluor 555–EGF (20 ng ml−1) for the indicated times, fixed and examined by fluorescence microscopy. Data are representative of three independent experiments. (e) ChIP assay for the binding of HIF-1α to the RABEP1 promoter in HEK293 cells transfected with PLD1-PH. (f) Quantitative RT–PCR assay of rabaptin-5 mRNA in HEK293 cells transfected with GFP–PLD1-PH and incubated under hypoxia. *P<0.05, results are shown as the mean±s.d. of the three independent experiments. EGFR, epidermal growth factor receptor; HIF-1α, hypoxia-inducible factor-1α GFP, green fluorescent protein; PH, pleckstrin homology; PLD, phospholipase D.
Figure 5
Figure 5
PLD1-PH attenuates tumor progression. (a) RT–PCR assay of rabaptin-5 mRNA in HT29 cells stably transfected with PLD1-PH. (b) HT29 cells expressing empty vector or PLD1-PH cells were xenografted on the left and right flanks of nude mice, respectively (arrows), then analyzed for tumor volume. Photographs show the representative tumors. Data were expressed as the mean±s.d. of seven different mice. (c) Immunoblot analysis of rabaptin-5 in tumor tissues from the xenografted mice injected with vector and PLD1-PH-expressed HT29 cells. Data are representative of three independent experiments. (d) Immunohistochemical staining of rabaptin-5 in the tumor tissues of xenografted mice injected with vector and PLD1-PH-expressed HT29 cells. Data are representative of three independent experiments. PH, pleckstrin homology; PLD, phospholipase D; RT–PCR, reverse transcription–PCR.

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