Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2

Abstract

The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1.

Figure 1.

COMP-Ang1 induces tyrosine phosphorylation of Tie1 in endothelial cells. (A and B) Isolated BECs (A) and LECs (B) were starved overnight and stimulated with COMP-Ang1 for 15 min or left unstimulated. The cell lysates were immunoprecipitated with anti-Tie1 or anti-Tie2 antibodies. Aliquots of the immunoprecipitates were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine, anti-Tie1, or anti-Tie2 antibodies. Relative mobilities of the molecular mass markers are indicated in kilodaltons. (C) HMEC-1 cells were stimulated with COMP-Ang1 for the indicated times followed by analysis of Tie1 and Tie2 as explained in A. The chemiluminescence signal was quantitated using Fluorchem digital imaging system (Flowgen Bioscience). Shown is relative phosphotyrosine intensity normalized to Tie1 or Tie2 protein levels. (D) HMEC-1 cells were stimulated with indicated amounts of COMP-Ang1 for 30 min followed by analysis of Tie1 and Tie2 as in C. The results are shown as a percentage of maximal receptor phosphorylation.

Figure 2.

Soluble Tie2 receptor inhibits COMP-Ang1–induced Tie1 phosphorylation. (A) EA.hy926 cells were stimulated with COMP-Ang1 or Ang2 or left unstimulated, followed by immunoprecipitation and analysis of Tie1 and Tie2, as in Fig. 1. White line indicates that intervening lanes have been spliced out. (B) EA.hy926 cells were stimulated with COMP-Ang1 alone or in combination with 10 μg/ml of soluble Tie1-Fc or Tie2-Fc for 15 min. As a control, cells were treated with soluble Tie receptors only. Tie1 and Tie2 were analyzed as in Fig. 1.

Figure 3.

COMP-Ang1–induced Tie1 phosphorylation is enhanced by Tie2. (A) The expression of human Ang1, Ang2, Tie1, and Tie2 was analyzed using Northern blotting of total RNA from the cell lines indicated. White lines indicate that intervening lanes have been spliced out. (B) 293T cells were transfected with Tie1 expression vector, as indicated, and stimulated or not with COMP-Ang1. Tie1 was analyzed as in Fig. 1. (C) 293 cells were transfected with expression plasmids for Tie1 and Tie2, as indicated, followed by analysis of Tie1 and Tie2 as in Fig. 1. (D) 293T cells were transfected with expression plasmids for wild-type or kinase-inactive Tie1 (K870R-Tie1) and Tie2, as indicated, followed by analysis of Tie1 as in Fig. 1. (E) 293 cells were transfected with expression plasmids for Tie1 and wild-type or kinase-inactive Tie2 (K855R-Tie2), followed by analysis of Tie1 as in Fig. 1.

Figure 4.

Tie1 and Tie2 interact at the cell surface. (A) 293T cells were transfected with Tie1-V5 or Tie2-Myc expression plasmids, as indicated. The cells were stimulated with COMP-Ang1 or left unstimulated, followed by chemical cross-linking of the cell surface proteins with DTSSP. Tie1 was immunoprecipitated using anti-V5 antibodies and analyzed as in Fig.1. (B) HUVECs were treated with control vehicle or DTSSP, followed by Tie2 immunoprecipitation. The immunoprecipitates and cell lysates were separated in SDS-PAGE and immunoblotted with anti-Tie2 and anti-Tie1 antibodies.

Figure 5.

Native Ang1 and Ang4, but not Ang2 or Ang3, induce Tie1 phosphorylation. (A and B) EA.hy926 cells were stimulated with indicated amounts of COMP-Ang1, native Ang1, Ang2, Ang3, Ang4, or COMP-HFARP or left unstimulated, followed by immunoprecipitation and analysis as in Fig. 1.