ALK5 phosphorylation of the endoglin cytoplasmic domain regulates Smad1/5/8 signaling and endothelial cell migration

Abstract

Endoglin, an endothelial cell-specific transforming growth factor-beta (TGF-beta) superfamily coreceptor, has an essential role in angiogenesis. Endoglin-null mice have an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin result in the vascular disease hereditary hemorrhagic telangiectasia type I. Increased endoglin expression in the proliferating endothelium of tumors has been correlated with metastasis, tumor grade and decreased survival. Although endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta-responsive pathways, the activin receptor-like kinase 5 (ALK5)/Smad2/3 pathway and the activin receptor-like kinase 1 (ALK1)/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Here, we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by ALK5 in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to TGF-beta and endothelial cell migration, whereas loss of phosphorylation at both serines 646 and 649 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to bone morphogenetic protein-9. Taken together, these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.

Fig. 1.

ALK5 basally phosphorylates endoglin. HA-endoglin expressed in COS-7 cells (A) and endogenous endoglin in WT MEECs (B) were orthophosphate labeled in the presence of increasing concentrations of the ALK5 inhibitor, SB431542. (C) HA-endoglin was expressed in WT mink lung cells (Mv1Lu) and mink lung cells that do not express ALK5 (R1B). (D) WT MEECs were ³²P-orthophosphate labeled in the presence of 200 pM TGF-β in the presence or absence of the ALK5 inhibitor SB431542 or 500 pM BMP-9 in the presence or absence of the ALK5 inhibitor, SB431542. Endoglin phosphorylation was assessed by ³²P-orthoposphate labeling of cells, immunoprecipitation of the HA-tagged endoglin and detection by phosphorimager (A, B, C, and D, top). Expression of endoglin was detected by western blot analysis (A, B, C, and D, bottom).

Fig. 2.

Endoglin is phosphorylated by ALK5 on S646 and S649. Empty vector (EV), HA-endoglin, HA-Δ3 (Δ3), HA-Δ10 (Δ10), HA-Δ21 (Δ21) or HA-Δ34 (Δ34) were transiently coexpressed with HA-caALK5 in COS-7 cells (A). Empty vector (EV), HA-endoglin or mutants, HA-M1 (M1), HA-M2 (M2), HA-M3 (M3) or HA-M4 (M4) were transiently coexpressed with HA-caALK5 in COS-7 cells (B). Empty vector (EV), WT endoglin (End), HA-S646A (S646A), HA-T647A (T647A), HA-S649A (S649A) or HA-T650A (T650A) were transiently coexpressed with HA-caALK5 in COS-7 cells (C). Endoglin phosphorylation was assessed by ³²P-orthophosphate labeling of COS-7 cells, immunoprecipitation of the HA-tagged receptors and detection by phosphorimager (top). Expression of HA-caALK5 was detected by autophosphorylation (middle) and expression of endoglin was detected by western blot analysis (bottom). Data are representative of three independent experiments.

Fig. 3.

Loss of endoglin phosphorylation by ALK5 on serines 646 and 649 impairs ALK1 phosphorylation but not TβRII phosphorylation. (A) HA-endoglin or HA-endoglin with both serines 646 and 649 mutated to alanine (S6/9A) were transiently coexpressed with HA-caALK1, HA-caALK5 or HA-tagged TβRII (HA-TβRII) in COS-7 cells. (B) HA-endoglin or HA-endoglin with serine 646 mutated to alanine (S646A), serine 649 mutated to alanine (S649A) or both serines 646 and 649 mutated to alanine (S6/9A) were transiently expressed in endoglin−/− mouse embryonic endothelial cells (KO MEECs). Endoglin phosphorylation was assessed by ³²P-orthophosphate labeling of cells, immunoprecipitation of the HA-tagged receptors and detection by phosphorimager (top). Expression of the kinases was detected by their autophosphorylation (top) and expression of endoglin was detected by western blot analysis (bottom). Data are representative of three independent experiments.

Fig. 4.

ALK1 does not phosphorylate unmodified endoglin. (A) HA-tagged kinase-dead ALK1 (kdALK1) or HA-tagged constitutively active ALK1 (caALK1) and HA-tagged kinase dead ALK5 (kdALK5) or HA-tagged constitutively active ALK5 (caALK5) were immunoprecipitated from COS-7 cells transiently expressing these constructs. The immunocomplexes were incubated with a GST fusion protein of the cytoplasmic domain of endoglin (GST-endoglin cyto) and ³²P-ATP, the GST-endoglin cyto was pulled down with glutathione beads and detected by phosphorimager (A, top). Expression of HA-endoglin, HA-caALK1 and HA-caALK5 was detected by western blot analysis (A, bottom). (B) HA-endoglin was expressed in COS-7 cells in the presence of empty vector or caALK1 and treated with the ALK5 inhibitor, SB431542. Endoglin phosphorylation was assessed by ³²P-orthoposphate labeling of cells, immunoprecipitation of the HA-tagged endoglin and detection by phosphorimager (top). Expression of HA-caALK1 was detected by phosphorimager (middle). Expression of endoglin was detected by western blot analysis (bottom). Data are representative of three independent experiments.

Fig. 5.

Phosphorylation of endoglin on S646 and S649 is required for induction of Smad1/5/8 phosphorylation. WT MEECs and KO MEECs expressing empty vector (EV), WT endoglin or endoglin mutants S646A, S649A or S6/9A were treated with 100 pM TGF-β1 (A) or 0.5 nM BMP-9 (B). Smad1/5/8 phosphorylation and total Smad levels were assessed by western blot analysis. Asterisk denotes non-specific band. Data are representative of three independent experiments.

Fig. 6.

Phosphorylation of endoglin on Ser646 regulates endothelial cell migration. WT MEECs and KO MEECs expressing empty vector (neo), HA-tagged WT endoglin (end) or endoglin mutants S646A or S649A (A) were plated on transwells coated with 50 mg/ml fibronectin and assessed for migration 6 h later (B). Cells were fixed and stained for their nuclei, and sample images were obtained. The number of migrated cells was counted and normalized to WT-neo MEECs and graphed (C). Expression of endoglin was assessed by cell surface biotinylation, immunoprecipitation and detection by streptavidin–horseradish peroxidase (A). Data are representative of four independent transwell migration experiments.