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. 2011 Jun 15;124(Pt 12):2021-31.
doi: 10.1242/jcs.075309. Epub 2011 May 24.

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

Natalia G Sampaio  1 , Wenfeng Yu, Dianne Cox, Jeffrey Wyckoff, John Condeelis, E Richard Stanley, Fiona J Pixley
Affiliations

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

Natalia G Sampaio et al. J Cell Sci. .

Abstract

Colony 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) γ2, and induced polarized PtdIns(3,4,5)P₃ 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.

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Figures

Fig. 1.
Fig. 1.
M−/−.Y721F macrophages display altered morphology. SEM images of M−/−.WT and M−/−.Y721F macrophages plated onto fibronectin-coated coverslips for 2 days and either kept in the continuous presence of CSF-1 (A) or incubated without growth factor overnight prior to stimulation with 120 ng/ml CSF-1 for 3 minutes (E) before fixation. Scale bars: 20 μM. M−/−.WT and M−/−.Y721F macrophages plated onto fibronectin-coated coverslips and grown in the continuous presence of CSF-1 were also fixed and stained for F-actin before cell outline tracing to measure the cell footprint area (B) and elongation ratio (maximum length:maximum width) (C). Data are means±s.e.m. (n≥40). ***P<0.001. (D) Forward scatter analysis of M−/−.WT and M−/−.Y721F cell size (FACSCantoII, BD Biosciences).
Fig. 2.
Fig. 2.
Paxillin and Pyk2 phosphorylation, and the incorporation of paxillin into adhesion structures, are reduced in M−/−.Y721F macrophages. (A) M−/−.WT and M−/−.Y721F macrophages were plated onto fibronectin-coated coverslips and incubated without growth factor overnight before stimulation with 120 ng/ml CSF-1 for 15 minutes followed by fixation, staining and examination by TIRF microscopy for adhesion structures rich in paxillin Y118-P. Scale bar: 20 μM. Lysates from CSF-1-starved M−/−.WT and M−/−.Y721F macrophages stimulated with 120 ng/ml CSF-1 for the indicated times were subjected to paxillin (B) or Pyk2 (C) immunoprecipitation (IP) then immunoblotted (WB) for the indicated proteins.
Fig. 3.
Fig. 3.
Loss of CSF-1R Y721-P signaling increases expression of the adhesion-regulating PTPϕ. (A) SEM images of M−/−.WT macrophages grown in the continuous presence of either GM-CSF or CSF-1 for 1 week before plating on fibronectin-coated coverslips for 2 days. Scale bar: 20 μM. (B) M−/−.WT and M−/−.Y721F cell lysates were examined by SDS-PAGE and immunoblotting for the indicated proteins. (C) Levels of mRNA encoding FAK, Pyk2 and PTPϕ were examined in M−/−.WT and M−/−.Y721F macrophages cultured in the continuous presence of CSF-1 by qRT-PCR analysis. Results are expressed as fold-change in M−/−Y721F cell gene expression compared with that in M−/−.WT cells. The data are means±s.e.m. for three independent experiments. *P<0.05.
Fig. 4.
Fig. 4.
CSF-1R Y721-P signaling regulates macrophage spreading, motility and promotion of carcinoma cell invasion. CSF-1-stimulated M−/−.WT (▴) and M−/−.Y721F (▪) macrophages plated onto glass-bottomed dishes were examined by time-lapse video microscopy and their cell outlines traced. The mean (±s.e.m.) total footprint area (A) and relative (%) increase in footprint area (B) for 30 minutes after CSF-1 (120 ng/ml) addition is shown (n≥40). (C) Total path length was measured by centroid analysis at 2.5-minute intervals up to 1 hour after CSF-1 stimulation and means±s.e.m. calculated for 10 cells each. (D) Boyden chamber analysis of CSF-1-stimulated chemokinetic (C/K) and chemotactic (C/T) motility is shown. The data are normalized to the M−/−.WT C/K result and are means±s.e.m. for a representative experiment (n=3). (E) M−/−.WT and M−/−.Y721F cells labeled with Cell Tracker Red (red) were injected into GFP-expressing MTLn3 xenograft carcinomas (green) 24 hours prior to imaging by multiphoton microscopy for 45 minutes. Scale bar: 20 μM. Two M−/−.WT and M−/−.Y721F cell (arrows) outlines in the first (green) and last (red) frame were overlaid to evaluate cell motility in vivo. (F) CFP-expressing MTLn3 carcinoma cells were either cultured alone or co-cultured with M−/−.WT or M−/−.Y721F cells and their capacity to invade more than 20 μM into overlaying collagen after 24 hours measured. The data are means±s.e.m. **P<0.01; ***P<0.001.
Fig. 5.
Fig. 5.
Mutation of CSF-1R Y721 abrogates its association with p85 PI3K, but not PLCγ, and induces PI3K expression. CSF-1-starved M−/−.WT and M−/−.Y721F cells were stimulated with CSF-1 for the indicated times and lysates subjected to CSF-1R (A), p85 PI3K (B), PY100 (C), PLCγ2 (D) and PLCγ1 (E) immunoprecipitation (IP). Eluted proteins were subjected to SDS-PAGE and immunoblotting (WB) for the indicated proteins. WCL, whole-cell lysates. (F) qRT-PCR analysis of mRNA encoding p85 PI3K and PLCγ2 in M−/−.WT and M−/−.Y721F macrophages grown continuously in CSF-1. Results are the fold-change in M−/−Y721F cell gene expression compared with that in M−/−.WT cells (means±s.e.m. for three independent experiments). *P<0.05. (G) CSF-1-starved M−/−.WT cells were treated with either 100 nM wortmannin or DMSO for 30 minutes before CSF-1 stimulation for the indicated times. F-actin-rich ruffles were visualized with Alexa-Fluor-568–phalloidin and quantified as described in the Materials and Methods.
Fig. 6.
Fig. 6.
The CSF-1R Y721-P motif is necessary and sufficient for CSF-1-induced ruffling and chemotaxis, as well as association of p85 PI3K and CSF-1R. (A) SEM images of M−/−.WT, M−/−.Y721F and M−/−.Y3AB,Y721 macrophages plated onto fibronectin-coated coverslips. Cells were incubated without growth factor overnight before stimulation with 120 ng/ml CSF-1 for 3 minutes. Scale bar: 20 μM. Boyden chamber analysis of CSF-1-stimulated chemokinetic (C/K) and chemotactic (C/T) motility in M−/−.WT, M−/−.Y721F and M−/−.Y3AB,Y721 macrophages (B) or in M−/−.Y3AB and M−/−.Y3AB,Y721 macrophages (C). Data are means±s.e.m. for a representative experiment (n=3). **P<0.01; ***P<0.001. (D) CSF-1R immunoprecipitations (IP) of lysates from CSF-1-starved M−/−.WT, M−/−.Y721F, M−/−.Y544,Y559,Y807 and M−/−.Y3AB,Y721 cells, incubated without growth factor overnight before stimulation with 120 ng/ml CSF-1 for the indicated times, were examined for associated p85 PI3K following SDS-PAGE. The numbers below the blot indicate the relative levels of co-immunoprecipitated PI3K, as determined by densitometry.
Fig. 7.
Fig. 7.
PI3K association with the CSF-1R through Y721-P regulates Akt activation and PtdIns(3,4,5)P3 production. (A) M−/−.WT, M−/−.Y721F, M−/−.Y544,Y559,Y807 and M−/−.Y3AB,Y721 cells were stimulated with 120 ng/ml CSF-1 and their lysates examined for Akt S473 or T308 phosphorylation. (B) M−/−.WT, M−/−.Y721F and M−/−.Y3AB,Y721 cells plated onto fibronectin-coated coverslips were incubated without growth factor overnight before stimulation with 120 ng/ml CSF-1 for 0 or 15 seconds and were then subjected to simultaneous fixation and extraction with 3.7% paraformaldehyde, 0.1% glutaraldehyde and 0.15 mg/ml saponin to examine CSF-1-induced PtdIns(3,4,5)P3 production. Representative merged images consist of PtdIns(3,4,5)P3 (red) and phase-contrast (grayscale) images of M−/−.WT (i,iv), M−/−.Y721F (ii,v) and M−/−.Y3AB,Y721 (iii,vi) cells treated with CSF-1 for 0 (i–iii) or 15 (iv–vi) seconds. Arrows indicate PtdIns(3,4,5)P3-encircled vesicles and arrowheads point to CSF-1-induced PtdIns(3,4,5)P3 in lamellipodia. Images are representative of those in four independent experiments. Scale bar: 10 μM.
Fig. 8.
Fig. 8.
The Y721-P motif triggers signaling that regulates the first peak of actin polymerization and the association of an ~170 kDa phosphorylated protein with a Rac-GTP or Cdc42-GTP complex. (A) Relative (%) increase in cellular F-actin content in CSF-1-starved M−/−.WT, M−/−.Y721F and M−/−.Y3AB,Y721 cells stimulated with 120 ng/ml CSF-1 for either 30 seconds or 3 minutes. The data are means±s.e.m. (n=4). *P<0.05. (B) CSF-1-starved M−/−.WT cells were stimulated with 120 ng/ml CSF-1 for the indicated times and lysates subjected to GST–PAK–CRIB pull down for Rac-GTP and Cdc42-GTP followed by SDS-PAGE and immunoblotting for Rac and Cdc42. Positive and negative controls (Cont) were prepared and run at the same time. (C) CSF-1-starved M−/−.WT and M−/−.Y721F cells were stimulated with 120 ng/ml CSF-1 for 0 or 30 seconds and lysates (Lys) subjected to GST–PAK–CRIB pull down for Rac-GTP and Cdc42-GTP followed by SDS-PAGE and immunoblotting for the indicated proteins. The arrow indicates the ~170 kDa phosphorylated protein, seen in M−/−.WT samples only.
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