Abstract
Signaling activated by binding of the CXC motif chemokine ligand 12 (CXCL12) to its cognate G protein-coupled receptor (GPCR), chemokine CXC motif receptor 4 (CXCR4), is linked to metastatic disease. However, the mechanisms governing CXCR4 signaling remain poorly understood. Here, we show that endocytosis and early endosome antigen 1 (EEA1), which is part of the endosome fusion machinery, are required for CXCL12-mediated AKT Ser/Thr kinase (Akt) signaling selective for certain Akt substrates. Pharmacological inhibition of endocytosis partially attenuated CXCL12-induced phosphorylation of Akt, but not phosphorylation of ERK-1/2. Similarly, phosphorylation of Akt, but not ERK-1/2, stimulated by CXCL13, the cognate ligand for the chemokine receptor CXCR5, was also attenuated by inhibited endocytosis. Furthermore, siRNA-mediated depletion of the Rab5-effector EEA1, but not of adaptor protein, phosphotyrosine-interacting with PH domain and leucine zipper 1 (APPL1), partially attenuated Akt, but not ERK-1/2, phosphorylation promoted by CXCR4. Attenuation of Akt phosphorylation through inhibition of endocytosis or EEA1 depletion was associated with reduced signaling to Akt substrate forkhead box O1/3a but not the Akt substrates TSC complex subunit 2 or glycogen synthase kinase 3β. This suggested that endocytosis and endosomes govern discrete aspects of CXCR4- or CXCR5-mediated Akt signaling. Consistent with this hypothesis, depletion of EEA1 reduced the ability of CXCL12 to attenuate apoptosis in suspended, but not adherent, HeLa cells. Our results suggest a mechanism whereby compartmentalized chemokine-mediated Akt signaling from endosomes suppresses the cancer-related process known as anoikis. Targeting this signaling pathway may help inhibit metastatic cancer involving receptors such as CXCR4.
Highlights
Signaling activated by binding of the CXC motif chemokine ligand 12 (CXCL12) to its cognate G protein– coupled receptor (GPCR), chemokine CXC motif receptor 4 (CXCR4), is linked to metastatic disease
Sclerosis complex 2; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol[3,4,5]-trisphosphate; PDK1, 3-phosphoinositide– dependent protein kinase-1; mTORC2, mTOR complex 2; pAkt-Ser473, phosphorylation of AKT Ser/Thr kinase (Akt) at Ser473; FoxO, forkhead box O; GSK3, glycogen synthase kinase 3; EGF, epidermal growth factor; PARP, poly(ADP-ribose) polymerase; ESCRT, endosomal sorting complex required for transport; FAK, focal adhesion kinase; MEM, minimum essential medium; TBS, Tris-buffered saline; ANOVA, analysis of variance
HeLa cells were treated with dynasore, an inhibitor of dynamin, a GTPase that is required for endocytosis [18] and is often used to inhibit GPCR endocytosis [19, 20]
Summary
CXCL12 binding to its cognate receptor, CXCR4, activates many signaling pathways, including the ERK-1/2 and Akt signaling pathways [1, 2, 4]. To determine how broadly applicable the requirement for endocytosis is in chemokine receptor–instigated Akt activation, we examined CXCL13, the sole cognate ligand for the chemokine receptor CXCR5 [3] This receptor is expressed endogenously in HeLa cells [21], and treatment of HeLa cells with increasing doses of CXCL13 robustly promoted phosphorylation of Akt and ERK-1/2 (Fig. 2, A and quantified in B). Dynasore treatment of WEHI-231 cells significantly attenuated CXCL12- or CXCL13-instigated phosphorylation of Akt (Fig. 4, A and quantified in B) These data suggest that endocytosis-mediated Akt activation may be broadly applicable to chemokine receptors and cell types. Dynasore treatment significantly attenuated CXCL12- or CXCL13-instigated phosphorylation of FoxO1/3a but not TSC2 or GSK3 (Fig. 3, B and quantified in E, F, and G, respectively) Taken together, these data suggest that endocytosis specifies Akt substrate phosphorylation downstream of chemokine receptor signaling
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