Phosphorylation of CSF-1R Y721 mediates its association with PI3K to regulate macrophage motility and enhancement of tumor cell invasion

Abstract

SummaryColony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. M-/-.Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of M-/-.Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) 2, and induced polarized PtdIns(3,4,5)P 3 production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.

CSF-1 Receptor Signaling in Myeloid Cells

“…Signaling may also occur from internalized receptors (Huynh et al 2012). Many of the proteins in complexes with the rapidly tyrosine phosphorylated cellular proteins are cytoskeletal proteins (Yeung et al 1998;Yeung and Stanley 2003) and the short-term responses include extensive cytoskeletal remodeling (Boocock et al 1989;Pixley et al 2001;Chitu et al 2005;Sampaio et al 2011;Pixley 2012). Increased protein synthesis is detected as early as 15 min following CSF-1R stimulation and plateaus at 2 h (Tushinski and Stanley 1983).…”

“…Later responses include increased motility and chemotaxis (Pixley et al 2001;Chitu et al 2005;Sampaio et al 2011;Pixley 2012), altered gene expression leading to the entry of cells into S phase at 12 h (Tushinski and Stanley 1985), during which time cell-surface CSF-1R levels are down-regulated, but cell-surface CSF-1Rs are turning over rapidly and presumably signaling Yu et al 2012;reviewed in Sherr 1991;Yeung and Stanley 2003). The expression of CD11b (Yu et al 2008) and of chemokines, cytokines, and cell-surface markers of M2 polarization state, are also increased (Fleetwood et al 2007;Foucher et al 2013).…”

“…Cbl activation leads to CSF-1R multiubiquitination, conformational changes, increased phosphorylation, and internalization of receptor-ligand complexes (Baccarini et al 1991;Rohde et al 2004;Xiong et al 2011). Tyr-721 is necessary and sufficient on the YEF.Y544,559,807AB background, for mediating macrophage chemotaxis to CSF-1 through the PI3K pathway (Sampaio et al 2011). Mutations of KI tyrosines 706 or 721, or carboxyterminal Tyr-974, alter morphological responses (Yu et al 2008).…”

“…3C) (Kelley et al 1999;Murray et al 2000;Golden and Insogna 2004;Chang et al 2009). In macrophages, Akt can be activated directly through the CSF-1R pTyr721/PI3K pathway (Lee and States 2000;Sampaio et al 2011) and indirectly by ceramide-1-phosphate (C1P) (Gó mez- Muñoz et al , 2005Muñoz et al , 2010Steinbrecher et al 2004), or the Gab2/PI3K pathway (Lee and States Arrows indicate activation; black line-capped arrows, inhibition; gray line-capped arrows, late-phase inhibition; round-capped arrows, increased expression or concentration; diamond-capped arrows, dissociation; dotted arrows, partial contribution; gold spheres, phosphotyrosines; and silver spheres, serine/threonine phosphorylation. Numbered gold spheres indicate the mouse CSF-1R tyrosine residues required for activation of specific pathways.…”

“…5) (Pixley et al 2001;Sampaio et al 2011). CSF-1-stimulates a biphasic actin polymerization response that initially peaks at 30 sec of stimulation and is followed by a longer-lasting wave, peaking at 5-6 min (Sampaio et al 2011;Ishihara et al 2012).…”