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First published online December 20, 2007
doi: 10.1242/10.1242/jcs.022681

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Phosphorylation of vascular endothelial cadherin controls lymphocyte emigration

Patric Turowski^1,*, Roberta Martinelli¹, Rebecca Crawford^1,, David Wateridge^1,, Anna-Pia Papageorgiou¹, Maria Grazia Lampugnani², Alexander C. Gamp⁵, Dietmar Vestweber⁵, Peter Adamson^1,¶, Elisabetta Dejana^2,3,4 and John Greenwood^1,*

¹ Division of Cell Biology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK
² Mario Negri Institute for Pharmacological Research, University of Milan, 20139 Milan, Italy
³ IFOM-IEO Campus, Via Adamello 16, University of Milan, 20139 Milan, Italy
⁴ Department of Biomolecular Sciences and Biotechnologies, Faculty of Sciences, University of Milan, 20139 Milan, Italy
⁵ Max-Planck-Institute of Molecular Biomedicine, Röntgenstr. 20, 48149 Münster, Germany

View larger version (86K): [in this window] [in a new window]   Fig. 1. Tyrosine phosphorylation of VEC following ICAM1 crosslinking or adhesion of lymphocytes. (A) GPNT cells were grown to confluence, serum starved and ICAM1 crosslinked (XL) for the indicated times. Total protein extracts (50 µg) were analyzed by immunoblotting with antibodies against phosphorylated tyrosine. Blots were subsequently stripped and probed for β-catenin as loading control. Four proteins with apparent molecular masses of 220 kDa, 140 kDa, 94 kDa and 83 kDa [previously identified as cortactin (Durieu-Trautmann et al., 1994)] displayed clearly enhanced tyrosine phosphorylation and are indicated by filled arrowheads. Open arrowhead indicates the position of the IgG heavy chains of the crosslinking antibody. (B) Confluent GPNT cells were serum starved and either (a,m) left untreated or ICAM1 crosslinked for (b) 15 minutes or (c-l) the different times indicated. Cells were fixed, extracted and stained for (a,b) surface ICAM1, (c-g) phosphorylated tyrosine, (h-l) F-actin or (m) VEC. Bar, 10 µm. (C-F) Confluent (C,D,F) GPNT cells or (E) mouse brain endothelioma EC, bEND5, were serum starved and subjected to crosslinking of ICAM1 (XL) or unrelated surface molecules (MHC class I; endomucin, EMCN). At the indicated times cells were washed and lysed. VEC immunoprecipitates were then analyzed by immunoblotting using either antibodies against phosphorylated tyrosine or VEC. (D) The amount of tyrosine-phosphorylated VEC (see C) was quantified by densitometry from five independent experiments and expressed as fold-increase of untreated controls (mean ± s.e.m.). (F) Prior to ICAM1 crosslinking (15 minutes), and where indicated, cells were pre-treated using PP2 (10 µM, 30 minutes), C3 transferase (2 µg/ml, 16 hours), cytochalasin D (CD, 2 µM, 30 minutes) or BAPTA (BA, 20 µM, 30 minutes). (G,H) Confluent GPNT cells were co-cultured with rat peripheral lymph node (PLN) lymphocytes (approximately five lymphocytes per EC). At the indicated times cells were lysed and VEC immunoprecipitates prepared and analyzed as described above. (H) Data from four independent experiments were quantified by densitometry, normalized and expressed as fold-increase of untreated controls (mean ± s.e.m.). Significant differences were determined by Student's t-test (*P<0.003, **P<0.002). In all blots the position of size markers (in kDa) is indicated on the left.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Analysis of tyrosine phosphorylation of other junction proteins in ICAM1 stimulated ECs. (A-E) Confluent GPNT cells were serum starved and ICAM1 crosslinked (XL). At the indicated times cells were washed, lysed and subjected to immunoprecipitation of (A) ZO-1 and (B-E) catenins as indicated. Immunoprecipitates were then analyzed by immunoblotting using antibodies against phosphorylated tyrosine, ZO-1, VEC or catenin. Black and white arrowheads in A-E indicate the position of migration of VEC and relevant catenins, respectively, as determined by stripping and re-probing of the immunoblots. Immunoprecipitates of p120 did not contain detectable VEC, whether phosphorylated (black arrowhead) or not (data not shown). (F) The chicken occludin-expressing GPNT cell line (see supplementary material Fig. S1) was grown to confluence, serum starved and ICAM1 crosslinked (XL). At the indicated times chicken occludin was immunoprecipitated and analyzed by immunoblotting for phosphorylated tyrosine or occludin. In all blots the position of size markers (in kDa) is indicated on the left.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Conserved tyrosines in the cytoplasmic domain of VEC. (A) Sequence alignment of cytoplasmic domains of VEC from mouse, rat, human and chicken, and mouse E-cadherin. Tyrosines conserved in VEC and their relative position (mouse versus human) are indicated in green. Identical residues, conserved and semi-conserved substitutions are indicated by asterisks, colons and dots, respectively. Grey boxes correspond to those parts of E-cadherin that interact with β-catenin when crystallized together (Huber and Weis, 2001). (B) Mouse VEC was computer-modeled on the crystal structure of mouse E-cadherin in the E-cadherin–β-catenin complex (i.e. boxed in A) (pdb: 1i7x, 1i7w). Shown in the left panel is a ribbon representation of this model with the position and orientation of six tyrosines highlighted in green. The right panels represent enlarged views of areas surrounding these tyrosines. The β-catenin chain is in a space filling representation. Significantly, in this model, Y685, the residue predominantly phosphorylated following VEGF stimulation (Wallez et al., 2006), is not accessible when β-catenin is bound.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Y731 within the intracellular domain of VEC is important for lymphocyte migration. Mouse endothelioma cell lines, null for VEC and stably re-expressing wt or Y to F mutants of VEC were grown to confluence. (A) Equal amounts of proteins were analyzed by immunoblotting using anti-VEC and anti-ERK antibodies. The position of size markers (in kDa) is indicated on the left. (B) Immunocytochemical analysis of the VEC distribution. Bar, 10 µm. (C) Mouse endothelioma cell lines, null for VEC, stably re-expressing wt VEC or transiently nucleofected with the VEC-GFP-expressing plasmid pEGFP-N'-VEC were grown to confluence. They were then incubated with antigen-specific T cells, which were allowed to adhere and migrate for 4 hours. Adhesion (white) and migration (black) across these EC populations were then determined as described in the Materials and Methods section. Results are expressed as the percent increase of VEC-null EC (mean ± s.e.m. of six replicates from five independent experiments). (D) Lymphocyte migration across the indicated stable mouse endothelioma cell lines. Adhesion (white) and migration (black) across individual transfected EC populations were then measured as above. Results are expressed as % of control cells re-expressing wt VEC (mean ± s.e.m. of six replicates from at least three independent experiments). Significant differences were determined by Student's t-test (*P<0.005, **P<0.0001).

View larger version (112K): [in this window] [in a new window]   Fig. 5. Y to F substitutions in the intracellular domain of VEC at positions 645, 731 or 733 affect lymphocyte migration in a dominant manner. GPNT cells were nucleofected with wt or Y to F mutants of pEGFP-N'-VEC. On average 80% of cells expressed VEC-EGFP over a period of 3-4 days. (A) Transfected GPNT cells were fixed after 2 days and VEC-GFP distribution was analyzed by fluorescent microscopy. Bar, 50 µm. (B) Three days after transfection, GPNT cells were fixed and VEC-GFP was expression analyzed using confocal microscopy. Bar, 10 µm. (C) Nucleofected GPNT cells were grown to confluence for 24-48 hours at which point equal expression was verified by fluorescent microscopy (see A). Lymphocyte adhesion (white) and migration (black) were then measured as described in Fig. 4. (D) Mouse VEC-null endothelioma cells (see Fig. 4) were nucleofected with wt or the indicated Y to F mutants of pEGFP-N'-VEC before T cell adhesion and migration was assessed. Significant differences were determined by Student's t-test (*P<0.005; **P<0.0001).

View larger version (28K): [in this window] [in a new window]   Fig. 6. ICAM1 induced VEC phosphorylation in wt and mutant VEC. (A) CHO-ICAM1 cells were transfected with wt pEGFP-N'-VEC or not, grown to confluence and then starved. Cells were then subjected to ICAM1 crosslinking and VEC-GFP immunoprecipitated and analyzed by immunoblotting for phosphorylated tyrosine and VEC. C, untransfected controls; PV, sample from cells pretreated with pervandate (100 µM). (B) As described for A, except that the CHO-ICAM1 cells were transfected with wt or Y to F mutants of VEC as indicated. ICAM1 crosslinking was 10 minutes. (C) The sequence of the cytoplasmic domain of mouse VEC (as shown in Fig. 3A) has been used to predict tryptic peptides. Amino acids in small letters in peptide 11 are from the linker sequence to EGFP (which is not shown). Five out of the eleven peptides (bold) contain many phosphorylatable serine and tyrosine residues in line with our observation that VEC is strongly phosphorylated on serine and less so on tyrosine (data not shown). Note, in contrast to the report by Wallez et al. (Wallez et al., 2006) we have assumed that trypsin digestion does not occur when a proline is found at the carboxylic side of lysine or arginine. (D) CHO-ICAM1 cells were transfected with pEGFP-N'-VEC as described above. Cells were labeled with 32P and then subjected to ICAM1 crosslinking or not. VEC-GFP was immunoprecipitated and processed for tryptic peptide mapping. Arrows denote the position of crosslinking-specific phosphopeptides. The three maps displayed in a single row were chromatographed in the same tank and Rf values were directly comparable. Enlarged sections of the phosphopeptide maps showing ICAM1 crosslinking specific phosphopeptides are shown in supplementary material Fig. S2.

View larger version (28K): [in this window] [in a new window]   Fig. 7. ICAM1-mediated VEC phosphorylation affects paracellular migration and coincides with increased EC permeability. (A) GPNT cells were grown to confluence, serum starved and then either left untreated (NT) or subjected to ICAM1 crosslinking (XL), 50 ng/ml VEGF, 10 µM lysophosphatidic acid (LPA), 10 µM bradykinin (BK), 100 µM histamine (HST) or 1 U/ml thrombin (TBN) for 15 minutes. Subsequently, cells were lysed and VEC immunoprecipitates analyzed by immunoblots using anti-phosphorylated tyrosine or anti-VEC antibodies. (B) The flux of 4 kDa or 140 kDa FITC-dextran across confluent GPNT cell monolayers was measured when ICAM1 was crosslinked (XL) or not (NT). In each case, the FITC-dextran flux was linear over 120 minutes. The values shown are mean permeability changes that occurred over the initial linear 50-minute period following crosslinking in three independent experiments. (C,D) Confluent GPNT cells were serum starved and ICAM1 crosslinked (XL). At the indicated times cells were lysed and subjected to immunoprecipitation of VEC (C) or -catenin (D). Immunoprecipitates were then analyzed by immunoblotting using antibodies against phosphorylated tyrosine, -, β-, -catenins or VEC. Similar results were achieved when the order of the proteins for immunoprecipitates and immunoblots was inverted (data not shown). In all blots the position of size markers (in kDa) is indicated on the left. (E) GPNT cells were nucleofected with wt or Y to F mutants of pEGFP-N'-VEC as described in Fig. 3. They were then incubated with antigen-specific T cells, which were allowed to adhere and migrate for 1-4 h. Subsequently time-lapsed microscopy was performed over a period of 5-10 minutes to determine the fraction of T cells migrating in the paracellular area of the EC. Results are the mean ± s.e.m. of six replicates from at least three independent experiments. Significant differences were determined by Student's t-test (*P<0.05, **P=0.005, ***P<0.0001).