Interaction between endogenous LIMK1 and BMPR-II. (A) Schematic representation of BMPR-II, the short alternatively spliced isoform BMPR-IIs and the COOH-terminal tail construct, and recognition of these proteins, expressed in COS-7 cells as HA-tagged constructs, by anti–kinase domain and anti-tail domain antibodies. M, transmembrane region. (B) Recognition of endogenous BMPR-II by anti–BMPR-II-tail antibodies in Western immunoblotting of lysates from the indicated cell lines. (C) Association of endogenous BMPR-II and LIMK1 in NIH3T3 cells. Lysates from NIH3T3 cells were immunoprecipitated with anti–BMPR-II-tail antibodies, and the presence of BMPR-II and LIMK1 in the immunocomplexes was determined by Western immunoblotting with the indicated antibodies. Two percent of the total lysate used for immunoprecipitation was tested by immunoblotting for LIMK1 expression (Lysate). FLAG-tagged LIMK1 and HA-tagged BMPR-II immunoprecipitated from transfected COS-7 cells with antibodies against these epitopes served as marker controls.

Inhibition of LIMK1 function after interaction with BMPR-II. (A) In vitro kinase assay of coimmunoprecipitated myc-tagged LIMK1 and BMPR-II (M-LIMK1 and M-BMPR-II) proteins using 5 μg of GST–cofilin as substrate. Autophosphorylated M-LIMK1 and M-BMPR-II and phosphorylated GST–cofilin (arrowheads) and their level of expression as determined by immunoblotting (arrows) are indicated at the top and bottom panels, respectively. The level of cofilin phosphorylation is an indication of LIMK1 activity, and the fold change in cofilin phosphorylation was calculated by PhosphorImage analysis after normalization for the level of LIMK1 expression as determined by immunoblotting. The level of cofilin phosphorylation in the presence of LIMK1 alone was used as the baseline and designated 1.0. (B) In vitro kinase assays of coimmunoprecipitated FLAG- or GFP-tagged LIMK1 (F- or GFP-LIMK1) or GFP-tagged kinase domain of LIMK1 (GFP-KIN) with or without M-BMPR-II. (C) In vitro kinase assays of immunoprecipitated GFP–LIMK1 in the presence or absence of tailless kinase dead BMPR-II (BMPRII-T-KD). 5 μg of GST–cofilin is used as substrate in all samples. Phosphorylated proteins (arrowheads, top panel) and their level of expression after immunoblotting (arrows, bottom panel) are indicated. The fold change in GST–cofilin phosphorylation by LIMK1 was calculated as described above.

Schematic representation of two signaling outputs from the BMPR system. In the canonical Smad signaling process, BMP4 binds to and brings together BMPR-I (-IA or -IB, also known as ALK3 and ALK6, respectively) and BMPR-II. BMPR-II phosphorylates the regulatory region (green box) of BMPR-I, activating the kinase domain (blue box), which phosphorylates Smad1, leading to its nuclear translocation for regulation of target genes. The present results show that the COOH-terminal tail domain (yellow box) of BMPR-II in the basal state binds to the LIM domain region of LIMK1 and inhibits LIMK1. BMP4 binding relieves this inhibitory interaction, enabling LIMK1 to phosphorylate cofilin, thereby regulating the actin cytoskeleton.

Immunoprecipitation analyses of overexpressed LIMK1 and its interaction with BMPR-II proteins in COS-7 cells. (A) Schematic representation of full-length and truncated BMPR-II proteins. The extracellular domain (gray), the transmembrane and kinase domains (small and large black areas, respectively), and the cytoplasmic tail (white) are presented. The numbers of the amino acid residues are indicated above each structure as well as the site of the most COOH-terminal mutation currently identified in PPH patients, R873X. (B) Immunoprecipitation and immunoblot analyses of GFP-tagged LAPs interacting with FLAG-tagged LIMK1 (F-LIMK1) but not FLAG–Btk (F-Btk). (C) GST-tagged LIMK1 interaction with full-length myc-tagged BMPR-II (M-BMPR-II) or FLAG-tagged truncated BMPR-II (F-BMPR-II-T; contains no cytoplasmic tail).

Analysis of LIMK1 and BMPR-II interaction. (A) GST–LIMK1 interaction with myc-tagged LAP proteins (M-hLAP15s and M-mLAP16s); isolated regions of the cytoplasmic tail of BMPR-II. (B) GST–LIMK1 interaction with wild-type, untagged BMPR-II and mutated BMPR-II (B-R873X) containing a COOH-terminal mutation (see Fig. 1 A). (C) FLAG-tagged LIM and PDZ domains of LIMK1 (F-LIM1,2 and F-PDZ) and full-length F-LIMK1, but not FLAG-tagged LIMK1 kinase domain (F-KIN) or F-Btk, interact with full-length BMPR-II. A schematic diagram of the LIMK1 domains is represented below the panels. The LIM domains (dark gray), PDZ domain (black), and kinase region (light gray) are indicated. (D) Association of two different amounts (10 and 20 μl) of GST–LIMK1 bound to glutathione-Sepharose beads with HA–PAK4 (lanes 1 and 4), BMPR-II (lanes 2 and 5), and both PAK4 and BMPR-II (lanes 3 and 6). Numbers above the blots indicate the fold change in HA–PAK4's ability to bind GST–LIMK1 in the presence and absence of overexpressed BMPR-II.

Effects of BMP4 on LIMK1 activity and subcellular localization. (A) Immunoblot of COS cell lysates before and after stimulation with 10 ng/ml BMP4. The membrane was probed with anti–phospho-cofilin, stripped, and reprobed with anti-cofilin antibodies. The numbers below indicate the fold induction of phospho-cofilin level after BMP4 stimulation and were adjusted for the level of cofilin in the lysates. (B) Immunoblots of cell lysates prepared from COS cells overexpressing GST–LIMK1, BMPR-II, and both GST–LIMK1 and BMPR-II after or before BMP4 stimulation. The filters were probed with anti–BMPR-II (tail), anti-LIMK1 (rat monoclonal), anti–phospho-cofilin, and anti-cofilin antibodies. The numbers below indicate the fold induction of phospho-cofilin (P-cofilin) level after BMP4 stimulation and were adjusted for the level of cofilin in the lysates. The levels of overexpressed GST–LIMK1 and BMPR-II were consistently much higher when expressed separately than when coexpressed in the same cells. (C) Immunofluorescence analysis of endogenous LIMK1 and actin colocalization in unstimulated COS-7 cells (top) and in COS-7 cells stimulated with 100 ng/ml BMP4 for 10 min (middle and bottom). Arrowheads highlight the coredistribution of LIMK1 and F-actin to the cell's peripheral ruffles. Bar, 20 μM. (D) Immunohistochemical analysis of endogenous LIMK1 expression in the precapillary pulmonary artery of normal human lung (i) and in lung tissue from an individual with PPH (ii).