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. 2005 Nov;33(5):438-46.
doi: 10.1165/rcmb.2005-0103OC. Epub 2005 Jul 7.

Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension

Wai K P Wong  1 , James A Knowles, Jane H Morse
Affiliations

Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension

Wai K P Wong et al. Am J Respir Cell Mol Biol. 2005 Nov.

Abstract

Mutations of bone morphogenetic protein receptor type II (BMPR-II) have been associated with familial and idiopathic pulmonary arterial hypertension (PAH). BMPR-II is a member of the transforming growth factor-beta receptor superfamily. It consists of extracellular, transmembrane, and kinase domains, and a unique C-terminus with mostly unknown function. However, a number of PAH-causing mutations are predicted to truncate the C-terminus, suggesting that this domain plays an important role in the homeostasis of pulmonary vessels. In this study, we sought to elucidate the functional role of this C-terminus by seeking its interacting partners. Using yeast two-hybrid screening, we identified c-Src tyrosine kinase as a binding partner of this C-terminus. In vitro co-immunoprecipitation confirmed their interaction. Mutations truncating the C-terminus disrupted their interaction, while missense mutation within kinase domain reduced their interaction. In addition, BMPR-II and c-Src tyrosine kinase colocalized within intracellular aggregates when overexpressed in HEK293 cells. Moreover, mutations truncating the C-terminus disrupted their colocalization, whereas missense mutation within kinase domain had no effect on their colocalization. Furthermore, BMP ligand stimulation decreased c-Src-activating phosphorylation at Tyrosine 418 in pulmonary smooth muscle cells in both time- and concentration-dependent manners. Mutations that truncated the C-terminus abolished this response. Taken together, these results suggest a model in which proliferative effect of c-Src by vasoactive molecules is balanced by opposing effect of BMP signaling in basal state, and the loss of this balance due to BMPR2 mutations leads to increased c-Src activity and subsequently cell growth.

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Figures

<b>Figure 1.</b>
Figure 1.
Isolation of protein interacting with C-terminal domain of BMPR-II. (A) Growth of yeast transformants on YPD and high-stringency selection plates. One colony of each of the indicated yeast transformants was resuspended in 0.5 ml of water and streaked with three repeats onto rich yeast peptone dextrose (YPD) medium (left panel) or high-stringency selection plates containing X-α-Gal (right panel). Yeast cells were transformed with the following plasmids: (1) the C-terminal domain of BMPR-II (pGBKT7-DBD-BMPR-II [575–1038]) and the isolated positive library clone (pGADT7-library clone-AD) from the yeast two-hybrid screening; (2) C-terminal domain of BMPR-II and pGADT7 empty vector; and (3) pGBKT7-DBD empty vector and the isolated library clone. (B) Schematic representation of the library clone isolated from the yeast two-hybrid screening. The isolated library clone was identified by direct sequencing and consisted of partial sequence of the full-length c-Src tyrosine kinase. The numbers of the amino acid residues were indicated. The isolated library clone consisted of the Src Homology (SH) domains 2 and 3, and the catalytic tyrosine kinase.
<b>Figure 2.</b>
Figure 2.
Co-immunoprecipitation of BMPR-II with c-Src tyrosine kinase. (A) Schematic representation of expression constructs of wild-type, truncation, and point mutants of BMPR-II. The number of amino acids of BMPR-II and its various domains were indicated: ECD (extracellular domain), TD (transmembrane domain), Kinase, and C-terminal domain. All of the expression constructs were N-terminal tagged with 6xHIS. (B) Co-immunoprecipitation of BMPR-II with c-Src tyrosine kinase. The HEK293 cells were transfected with the plasmid encoding HA-tagged c-Src tyrosine kinase and without (lane 1) or with the plasmid encoding 6xHIS-tagged wild-type BMPR-II (lane 2) or with various mutant BMPR-IIs (lanes 3–5). The cell lysates were immunoprecipitated with anti-HIS antibody and immunoblotted with anti-HA antibody to detect for the presence of c-Src tyrosine kinase.
<b>Figure 2.</b>
Figure 2.
Co-immunoprecipitation of BMPR-II with c-Src tyrosine kinase. (A) Schematic representation of expression constructs of wild-type, truncation, and point mutants of BMPR-II. The number of amino acids of BMPR-II and its various domains were indicated: ECD (extracellular domain), TD (transmembrane domain), Kinase, and C-terminal domain. All of the expression constructs were N-terminal tagged with 6xHIS. (B) Co-immunoprecipitation of BMPR-II with c-Src tyrosine kinase. The HEK293 cells were transfected with the plasmid encoding HA-tagged c-Src tyrosine kinase and without (lane 1) or with the plasmid encoding 6xHIS-tagged wild-type BMPR-II (lane 2) or with various mutant BMPR-IIs (lanes 3–5). The cell lysates were immunoprecipitated with anti-HIS antibody and immunoblotted with anti-HA antibody to detect for the presence of c-Src tyrosine kinase.
<b>Figure 3.</b>
Figure 3.
Recruitment and co-localization of c-Src tyrosine kinase with BMPR-II. (A) Confocal images of negative control (without primary antibody). (B) 6xHIS-BMPR-II expressed alone. HEK293 cells were transfected with wild-type BMPR-II, permeablilized, and stained with antibody against 6xHIS and FITC-conjugate anti-HIS antibody to visualize BMPR-II (green). (C) HA–c-Src tyrosine kinase expressed alone. Cells were detected by using antibody against HA and rhodamine-conjugated monoclonal anti-HA antibody to visualize c-Src tyrosine kinase (red). (D–F) Confocal images of (D) 6xHIS-BMPR-II and (E) HA-c-Src tyrosine kinase in the same HEK293 cell overexpressing both BMPR-II and c-Src tyrosine kinase. (F) 6xHIS-BMPR-II colocalized with HA-c-Src tyrosine kinase in intracellular vesicle-like aggregates (indicated by arrows) in the merged image. Data were representative of three independent experiments.
<b>Figure 4.</b>
Figure 4.
Effects of PAH mutations on the colocalization of BMPR-II and c-Src tyrosine kinase in HEK293 cells. (A–C) HEK293 cells were transiently transfected with HA–c-Src tyrosine kinase and truncation mutant BMPR-II (K230fsX21), permeablilized, and stained with antibody against 6xHIS and FITC-conjugate anti-HIS antibody to visualize BMPR-II (K230fsX21) (green) and with antibody against HA and rhodamine-conjugated monoclonal anti-HA antibody to visualize c-Src tyrosine kinase (red). Merged image depicted lack of colocalization of BMPR-II (K230fsX21) with c-Src tyrosine kinase. (D–F) HEK293 cells expressing BMPR-II (R491W) point mutant and c-Src tyrosine kinase. Colocalization of BMPR-II point mutant and c-Src tyrosine kinase was found in the merged image (aggregates indicated by arrows). (G–I) Similar to the K230fsX21 BMPR-II mutant, lack of colocalization between BMPR-II and c-Src tyrosine kinase was also found with N861fsX10 BMPR-II mutant. Data were representative of three independent experiments.
<b>Figure 5.</b>
Figure 5.
BMP-2, -4, and -7 stimulations inhibit c-Src phosphorylation (Tyr418). The human pulmonary smooth muscle cells were stimulated with 200 ng/ml of BMP-2, BMP-4, BMP-7 or without ligand as control for the indicated time periods. After ligand stimulation, cells were immediately fixed with 4% formaldehyde in PBS and assayed for the phosphorylation of c-Src (Tyr418). Results were normalized with the number of cells as described in MATERIALS AND METHODS. Data represented the mean ± SEM from two independent experiments with duplicates.
<b>Figure 6.</b>
Figure 6.
BMP-2 exposure results in concentration-dependent decrease of c-Src phosphorylation (Tyr418). The human pulmonary smooth muscle cells were plated and treated with various concentrations of BMP-2. Cells were then fixed and assayed for the phosphorylation of c-Src (Tyr418). Results were normalized with the number of cells. Data represented the mean ± SEM from two independent experiments with duplicates.
<b>Figure 7.</b>
Figure 7.
Effects of BMPR-II mutations on c-Src phosphorylation (Tyr418). The human pulmonary arterial smooth muscle cells were transfected with BMPR-II wild-type or mutant (K230fsX21, N861fsX10, or R491W) expression constructs or empty vector (as control). Forty-eight hours after transfection, cells were treated with or without BMP-2 for 10 min. The cells were then fixed and assayed for c-Src phosphorylation (Tyr418). Results were normalized with the number of cells. Data represented the mean ± SEM from two independent experiments with duplicates.
<b>Figure 8.</b>
Figure 8.
Schematic representation of c-Src tyrosine kinase as a potential hub for signaling pathways involved in PAH. The interaction between BMPR-II and c-Src tyrosine kinase may inhibit c-Src tyrosine kinase activity in the presence of BMP ligand by reducing its phosphorylation at tyrosine-418 residue. The inhibition of c-Src activity by BMP signaling may inhibit downstream cell cycle regulators such as cyclins D and E and subsequently prevent smooth muscle cell proliferation. Moreover, BMP ligand stimulation may also inhibit cell proliferation through the Smad pathway. In contrast, activation of c-Src tyrosine kinase by 5-HT2B receptor causes the induction of cell cycle regulators (cyclins D and E) via ERK/MAPK pathway and subsequently triggers cell proliferation. In addition, activation of c-Src tyrosine kinase by serotonin inhibits voltage-dependent and Ca2+-activated K+ channel (MaxiK, BK), leading to vasoconstriction. Protein–protein interaction between c-Src tyrosine kinase and voltage-gated K+ channel (Kv1.5) suppresses outward potassium current and ultimately causes vasoconstriction. In contrast, activation of c-Src tyrosine kinase by angiotensin-II (Ang-II) receptor leads to increased eNOS gene expression and activities via MAPK pathway and subsequently causes vasorelaxation. The “+” and “−” signs indicated possible activation and inhibition, respectively, while “?” indicated potential cross-talk of the smad and c-Src signaling pathway.
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