CXCL12-induced macropinocytosis modulates two distinct pathways to activate mTORC1 in macrophages.

Although growth factors and chemokines elicit different overall effects on cells-growth and chemotaxis, respectively-and activate distinct classes of cell-surface receptors, nonetheless, they trigger similar cellular activities and signaling pathways. The growth factor M-CSF and the chemokine CXCL12 both stimulate the endocytic process of macropinocytosis, and both activate the mechanistic target of rapamycin complex 1 (mTORC1), a protein complex that regulates cell metabolism. Recent studies of signaling by M-CSF in macrophages identified a role for macropinocytosis in the activation of mTORC1, in which delivery of extracellular amino acids into lysosomes via macropinocytosis was required for activation of mTORC1. Here, we analyzed the regulation of macropinosome (MP) formation in response to CXCL12 and identified 2 roles for macropinocytosis in the activation of mTORC1. Within 5 min of adding CXCL12, murine macrophages increased ruffling, macropinocytosis and amino acid-dependent activation of mTORC1. Inhibitors of macropinocytosis blocked activation of mTORC1, and various isoform-specific inhibitors of type 1 PI3K and protein kinase C (PKC) showed similar patterns of inhibition of macropinocytosis and mTORC1 activity. However, unlike the response to M-CSF, Akt phosphorylation (pAkt) in response to CXCL12 required the actin cytoskeleton and the formation of macropinocytic cups. Quantitative fluorescence microscopy showed that phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a product of PI3K and an upstream activator of Akt, localized to macropinocytic cups and that pAkt occurred primarily in cups. These results indicate that CXCL12 activates mTORC1 via 2 mechanisms: 1) that the macropinocytic cup localizes Akt signaling and 2) that MPs convey extracellular nutrients to lysosomes.