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. 2008 Jul 1;314(11-12):2212-23.
doi: 10.1016/j.yexcr.2008.03.016. Epub 2008 Apr 7.

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells

E Merit Reyes-Reyes  1 , Steven K Akiyama
Affiliations

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells

E Merit Reyes-Reyes et al. Exp Cell Res. .

Abstract

We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell-surface nucleolin, PI3-K and p38 MAPK. Using siRNA approaches, we have found that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates.

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Figures

Figure 1
Figure 1. P-Selectin Glycoprotein Ligand 1 and CD24 are expressed by Colo-320 cells, but do not function as P-selectin receptors
(A) Total Colo-320 cell lysates (lane 1) and P-selectin ligands purified by affinity chromatography from Colo-320 cells (lane 2) were resolved by 4-12% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with either anti-P-selectin glycoprotein ligand 1 (PSGL-1) or anti-CD24 antibodies. (B and C) Colo-320 cells were incubated with either 10 μg/ml anti-CD24 antibody (Panel B, blue line), anti-PSGL-1 (Panel C, blue line), or 10 μg/ml non-immune mouse IgG (gray histograms), followed by FITC-conjugated anti-mouse IgG-Fc F(ab)2, and analyzed by flow cytometry.
Figure 2
Figure 2. Identification of nucleolin (or a nucleolin-like protein) as the major protein eluted from P-selectin affinity and as a cell surface protein in Colo-320 cells
Cell-surface proteins from intact Colo-320 cells were labeled covalently with membrane impermeable sulphosuccinimidobiotin. Biotinylated plasma membrane proteins were purified and then incubated with, and eluted from, either a P-selectin affinity column or a control IgG-Fc column. (A) Proteins eluted from P-selectin IgG-Fc affinity column (Psel) or IgG-Fc affinity column (Fc) were detected by blotting with HRP-conjugated streptavidin. (B) Nucleolin was immunoprecipitated from the proteins eluted from P-selectin-IgG-Fc affinity column (Psel) or IgG-Fc affinity column (Fc) and analyzed by blotting with either anti-nucleolin antibody (upper panel) or HRP-conjugated streptavidin (lower panel). The arrowhead indicates the band corresponding to nucleolin. (C) Protein G-coated microwells were incubated with 2% denatured BSA/PBS in absence or presence of 20 μg/ml anti-nucleolin antibodies (D3 or 4E2) or non-immune mouse IgG. Colo-320 cells (5×104 cells) in 2% denatured BSA/PBS were allowed to attach to antibodies immobilized for 30 min at 4°C. Error bars indicate standard deviation.
Figure 3
Figure 3. Cell-surface nucleolin functions as P-selectin binding protein in Colo-320 cells
(A) Colo-320 cells were allowed to attach to immobilized BSA (white bars) or P-selectin (black bars) substrates for 60 min in the absence (-) or presence of 20 μg/ml anti-nucleolin mAb (D3) or non-immune mouse IgG for 60 min at 37°C. Error bars indicate standard deviation. (B) Colo-320 cells were incubated with P-selectin IgG-Fc chimera at 4°C for 45 min. After washing, cell were plated on polylysine-coated cover slips in the presence or absence of anti-human IgG-Fc (cross-linker) and incubated for 10 min at 37°C, washed, and fixed. The localization of nucleolin on non-permeabilized cells was determined by staining with antibody against nucleolin and with Alexa 488 goat anti-mouse IgG-Fc (green). P-selectin was localized with Alexa 565 goat anti-human IgG-Fc (red). The nucleus was stained with DAPI (blue). Fluorescent images were combined to determine colocalization (yellow). Bar = 10 μM.
Figure 4
Figure 4. P-Selectin induces an increase of tyrosine phosphorylation of cell surface nucleolin
(A) Colo-320 cells were incubated in suspension without (lane 1) or with 20 μg/ml human-IgG (Fc) (lane 2) or P-, L-, or E-selectin-IgG-Fc (lanes 3, 4, and 5, respectively) for 5 min at 37°C. Cells were then lysed and nucleolin was immunoprecipitated and analyzed by immunoblotting with anti-phosphotyrosine antibody (upper panel). After stripping bound antibodies, the membrane was probed for total nucleolin (lower panel). The arrowheads indicate the positions of the nucleolin bands. (B) Cells incubated with or without 20 μg/ml P-selectin-IgG-Fc (Psel) for 5 min at 37°C were fractionated into membrane, cytoplasmic, and nuclear fractions. Nucleolin was immunoprecipitated from each cellular fraction and analyzed by immunoblotting with anti-phosphotyrosine antibody (upper panel). After stripping bound antibodies, the membrane was probed for total nucleolin (lower panel). (C) Total lysates from each cell fraction from panel B were analyzed by immunoblotting with antibodies for a plasma membrane marker (anti-Na+/K+-ATPase α1) and a nuclear marker (anti-histone 3) to show absence of cross-contamination of the membrane and nuclear fractions.
Figure 5
Figure 5. Nucleolin co-immunoprecipitates with p38 MAPK and PI 3-K on a P-selectin-dependent manner
Colo-320 cells were incubated in suspension without (-) or with 20 μg/ml P-selectin-IgG-Fc (Psel) for 5 min. Then, p38 MAPK, nucleolin, or PI 3-K p110 were immunoprecipitated. (A) p38 MAPK and nucleolin immunoprecipitates were analyzed by immunoblotting with anti-nucleolin, anti-phospho-p38 MAPK, and anti-total p38 MAPK. (B) PI 3-K and nucleolin immunoprecipitates were analyzed by immunoblotting with anti-nucleolin and anti- PI 3-K (p85). (C) Cells were pretreated with or without 200 μM genistein for 30 min at 37°C prior to P-selectin stimulation. Nucleolin immunoprecipitates from treated and untreated cell lysates were analyzed by immunoblotting for phosphotyrosine, p38 MAPK, or PI3-K. After stripping bound antibodies, the same membranes were reprobed for total nucleolin. The arrowhead indicates the nucleolin band.
Figure 6
Figure 6. Nucleolin expression is required for formation of the P-selectin-mediated PI 3-K/p38 MAPK signal complex formation
Colo-320 cells were transfected with nucleolin siRNAs (NCL), control siRNAs (C), or incubated under the same conditions in the presence (M) or absence (B) of transfection reagent. After culturing the cells for 48 h after transfection, (A) cells were lysed and analyzed by immunoblotting with anti-nucleolin antibody. After stripping bound antibodies, the same membranes were reprobed for GAPDH (lower panels). (B) Transfected Colo-320 cells were incubated with 20 μg/ml P-selectin-IgG-Fc or with 20 μg/ml human IgG-Fc, followed by FITC-conjugated anti-human IgG-Fc F(ab)2, and binding was assessed by flow cytometry. The % P-Selectin binding was calculated using the following formula: % P-selectin binding =Mean fluorescence (PselX-FcX)/ (PselBuffer-FcBuffer)*100, X= Transfected cells with nucleolin siRNAs, control siRNAs, or only incubated with presence (Mock) or absence (Buffer). (C) p38 MAPK was immunoprecipitated from transfected Colo-320 cells stimulated with P-selectin (Ps) or control, unstimulated (-), cells. p38 MAPK immunoprecipitates were analyzed by immunoblotting with anti-PI 3-K or anti-nucleolin. The membranes were probed for total p38 MAPK (lower panels) after stripping bound antibodies.
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