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Figure 5


Fig. 5. Binding of HUVECs to collagen via integrin {alpha}1 activates TCPTP and attenuates VEGFR2 signalling in endothelial cells. (A) Confocal-microscopy images from three-colour immunofluorescence stainings show the localization of integrin {alpha}1β1, vinculin and TCPTP in membrane protrusions in HUVECs adhering to collagen IV but not to gelatin. Scale bars: 10 µm. (B) Phosphatase activities (mean ± s.d.; n=3) were measured in control (IgG), TCPTP and SHP2 immunoprecipitates from HUVECs grown on collagen I or gelatin. The immunoprecipitates were also resolved on SDS-PAGE gels and probed for SHP2 and TCPTP. The phosphatase activities of TCPTP and SHP2 were normalized to the amounts of immunoprecipitated (IP) molecules. A representative experiment out of three with similar results is shown (mean ± s.e.m.; n=9; *P<0.05; ***P<0.005; n.s., not significant). (C) Serum-starved subconfluent HUVECs were treated for 1 hour with the integrin-{alpha}1 cytoplasmic-tail fusion peptide ({alpha}1-TAT, 200 nM) followed by VEGF-induction (100 ng/ml, 15 minutes) as indicated. Cell lysates were resolved on SDS-PAGE and immunoblotted for phosphorylated VEGFR2 (or tubulin as a control). (D) HUVECs were treated as in C and VEGFR2 kinase activity (mean ± s.d.; n=3) was measured from the cell lysates. **P<0.01. (E) Recombinant, purified TCPTP was incubated with no peptide (–), {alpha}1 or {alpha}2 cytoplasmic tail peptides (L amino acids), or with {alpha}1 and scrambled TAT peptides (D amino acids) and analyzed for phosphatase activity (mean ± s.d., n=3). **P<0.01.





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