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, 24 (18), 3235-46

Phosphorylation of EEA1 by p38 MAP Kinase Regulates Mu Opioid Receptor Endocytosis

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Phosphorylation of EEA1 by p38 MAP Kinase Regulates Mu Opioid Receptor Endocytosis

Gaëtane Macé et al. EMBO J.

Abstract

Morphine analgesic properties and side effects such as tolerance are mediated by the mu opioid receptor (MOR) whose endocytosis is considered of primary importance for opioid pharmacological effects. Here, we show that p38 mitogen-activated protein kinase (MAPK) activation is required for MOR endocytosis and sufficient to trigger its constitutive internalization in the absence of agonist. Further studies established a functional link between p38 MAPK and the small GTPase Rab5, a key regulator of endocytosis. Expression of an activated mutant of Rab5 stimulated endocytosis of MOR ligand-independently in wild-type but not in p38alpha-/- cells. We found that p38alpha can phosphorylate the Rab5 effectors EEA1 and Rabenosyn-5 on Thr-1392 and Ser-215, respectively, and these phosphorylation events regulate the recruitment of EEA1 and Rabenosyn-5 to membranes. Moreover, phosphomimetic mutation of Thr-1392 in EEA1 can bypass the requirement for p38alpha in MOR endocytosis. Our results highlight a novel mechanism whereby p38 MAPK regulates receptor endocytosis under physiological conditions via phosphorylation of Rab5 effectors.

Figures

Figure 1
Figure 1
p38 MAPK activation is necessary and sufficient for MOR endocytosis. (A) Western blots of lysates from HEK293 cells transfected with GFP-MOR or the vector, either unstimulated (C) or after stimulation with Damgo for 5 min (D) or UV. (B) Kinase activity in p38α immunoprecipitates from HEK293 cells transfected or not with GFP-MOR (+ and −, respectively) and either left untreated (Control) or stimulated with UV or Damgo. (C) Plasma membrane-associated GFP-MOR in HEK293 cells, either expressing MKK6DD or incubated with Damgo for 30 min in the presence or absence of SB203580, as indicated. GFP-MOR was detected by avidin pulldown of biotinylated membrane proteins followed by Western blot using a GFP antibody. The histogram represents the quantification of the GFP-MOR band (pulldown versus total cell lysate) using the Odyssey Imaging system. The experiment was repeated three times. (D) Damgo binding assays in wt or p38α−/− MEFs and p38α−/− MEFs expressing exogenous p38α, either unstimulated, after 30 min Damgo stimulation or transfected with MKK6DD. Results are expressed as the percentage of binding in wt MEFs and represent the mean±s.e.m. of six experiments. (E) Western blots of lysates from HEK293 cells expressing GFP-MOR alone (vector) or together with MKK6DD before (−) and after (+) 5 min of Damgo stimulation. The arrow indicates the overexpressed MKK6DD. (F) Western blots of lysates from wt and p38α−/− immortalized MEFs. (G) Western blots of lysates from wt and p38α−/− MEFs either transfected or not with MKK6DD (indicated by an arrow).
Figure 2
Figure 2
Specific activation of p38 MAPK stimulates morphine-induced MOR endocytosis. (A) Damgo binding assays in HEK293 cells expressing GFP-MOR alone (Vector) or together with MKK6DD, either unstimulated (Control) or after 30 min Damgo or morphine stimulation. The results are expressed as the percentage of binding in the untreated cells expressing GFP-MOR. The experiment was repeated three times. (B) Western blots of lysates from HEK293 cells expressing GFP-MOR alone or together with MKK6DD, nonstimulated (C) and stimulated with Damgo for 5 min (D) or with morphine for the indicated times. (C) Plasma membrane-associated levels of MOR in SH-SY5Y cells treated with Damgo or morphine, either alone or together with H2O2 in the presence or absence of SB203580, as indicated. MOR was visualized as in Figure 1C, but using a MOR antibody for the Western blot. The experiment was repeated twice.
Figure 3
Figure 3
Rab5 is required for MOR endocytosis. (A) Damgo binding assays in HEK293 cells expressing GFP-MOR alone or together with MKK6DD and transfected with Rab5S34N or RN-tre before and after Damgo stimulation. The values are normalized to those of untreated GFP-MOR cells and are expressed as the mean±s.e.m. of three experiments. (B) Western blots of lysates from HEK293 cells expressing GFP-MOR alone (Vector) or together with the indicated proteins, before (−) and after (+) Damgo stimulation for 5 min. (C) Damgo binding assays of wt and p38α−/− MEFs transfected with Rab5Q79L and myc-tagged p38α or the vectors alone and then incubated with SB203580, as indicated. The results are expressed as the percentage of binding in the corresponding control-vector cells. The experiment was repeated three times. (D) Western blots of lysates from wt and p38α−/− MEFs transfected with Rab5Q79L, myc-tagged p38α or the vectors alone, before and after Damgo stimulation for 5 min, as indicated.
Figure 4
Figure 4
p38α MAPK phosphorylates EEA1 and Rabenosyn-5. (A) In vitro kinase assay using MKK6DD-activated p38α (+) or MKK6DD alone (−) and the GST-fused C-terminus of EEA1 (amino acids 1257–1411) wt or with the mutation T1392A (10 μg each) as substrates (left panels). Western blot using a phospho-Thr antibody of GST-EEA1 (1257–1411) wt or T1392A (1 μg) after phosphorylation with MKK6DD-activated p38α or MKK6DD alone (right panels). (B) Endogenous EEA1 was immunoprecipitated from MKK6DD-expressing HEK293 cells (left panel) or SH-SY5Y cells (right panel) and blotted with antibodies against EEA1 or phospho-Thr. (C) HEK293 or SH-SY5Y cells were treated as indicated and endogenous EEA1 was immunoprecipitated and blotted with antibodies against EEA1 or phospho-Thr. (D) In vitro kinase assay using the indicated amounts of MKK6DD-activated GST-p38α and either 10 μg GST-EEA1 (1257–1411) or 15 μg His-GDI. (E) In vitro kinase assay using MKK6DD-activated p38α and the GST-Rabenosyn-5 wt or with the mutation S215A (10 μg each). (F) Endogenous Rabenosyn-5 was immunoprecipitated from SH-SY5Y cells expressing MKK6DD and blotted with antibodies against Rabenosyn-5 or phospho-Ser.
Figure 5
Figure 5
Phosphorylation of Thr-1392 regulates EEA1 subcellular localization. (A) In vitro recruitment of 35S-labelled EEA1 (1257–1411) wt and T1392A to early endosomes in the absence or in the presence of Rab5:GDI complex or GDI alone. Proteins bound to endosomal membranes were detected by autoradiography and Western blot. Syntaxin-13 was used as a membrane marker. (B) MEFs (wt or p38α−/−) were transiently transfected with full-length EEA1 either wt, T1392A or T1392D, fixed and stained with antibodies to EEA1. Scale bar represents 20 μm. (C) Western blots of membrane preparations from wt or p38α−/− MEFs transiently transfected with full-length EEA1 either wt, T1392A or T1392D. The histogram represents the mean±s.e.m. of two experiments.
Figure 6
Figure 6
p38 MAPKs regulate EEA1 membrane localization. (A) Western blots of membrane preparations from wt or p38α−/− MEFs transfected with EEA1wt and treated with 1 μM Damgo or 10 μM SB203580 for 30 min and 1 h, respectively. The histogram represents the quantification of two experiments. (B) Western blots of total lysates and membrane preparations from SH-SY5Y cells treated with Damgo, SB203580 or both together as in (A). The histogram represents the quantification by densitometric analysis of the EEA1 and Rabenosyn-5 bands versus Syntaxin-13. The experiment was repeated three times. (C, D) Western blots of total lysates and membrane preparations from HeLa cells either transfected with luciferase siRNA (Control) and a mixture of p38α and p38β siRNAs (C) or incubated with 10 μM SB203580 for 18 h. The experiment was repeated twice.
Figure 7
Figure 7
EEA1 phosphorylation is required for MOR endocytosis. (A) Western blots of total lysates from wt and p38α−/− MEFs transfected with Rab5Q79L and full-length wt EEA1, or the mutants T1392A and T1392D, as indicated. (B) Damgo binding assays in wt and p38α−/− MEFs transfected with Rab5Q79L alone or together with EEA1 wt or the indicated mutants. The results are expressed as the percentage of binding in cells transfected with the empty plasmids and represent the mean±s.e.m. of two experiments performed in triplicate. (C) Plasma membrane-associated levels of MOR in p38α−/− and wt cells transfected with either empty vector, EEA1 wt or the T1392D and T1392A mutants and treated with Damgo or Morphine, as indicated. MOR was visualized by avidin pulldown of biotinylated membrane proteins followed by Western blot using a MOR antibody. The histogram represents the quantification of the MOR band (pulldown versus total cell lysate) using the Odyssey Imaging system. The experiment was repeated twice. (D) Western blots of total lysates from wt and p38α−/− MEFs transfected with wt EEA1 or the mutants T1392A and T1392D and stimulated with Damgo or Morphine, as indicated.

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