Limits...
Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1.

Foletta VC, Lim MA, Soosairajah J, Kelly AP, Stanley EG, Shannon M, He W, Das S, Massague J, Bernard O, Soosairaiah J - J. Cell Biol. (2003)

Bottom Line: Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4.A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1.This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade Parkville, Victoria 3050, Australia.

ABSTRACT
Bone morphogenetic proteins (BMPs) regulate multiple cellular processes, including cell differentiation and migration. Their signals are transduced by the kinase receptors BMPR-I and BMPR-II, leading to Smad transcription factor activation via BMPR-I. LIM kinase (LIMK) 1 is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, we isolated clones encompassing the tail region of BMPR-II. Although the BMPR-II tail is not involved in BMP signaling via Smad proteins, mutations truncating this domain are present in patients with primary pulmonary hypertension (PPH). Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4. A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1. This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.

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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.
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fig3: 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.

Mentions: Having established that the tail of BMPR-II was required for its interaction with LIMK1, we then sought to define the minimal region within the tail required for this interaction. The sequences responsible for the LIMK1–BMPR-II interaction were further defined by creating myc-tagged BMPR-II tail deletion constructs. Plasmids encoding truncated versions of hLAP15 (hLAP15s, amino acids 742–932) and mLAP16 (mLAP16s, amino acids 742–1038) (Fig. 1 A) were cotransfected with constructs encoding either GST–LIMK1 or GST alone. After affinity purification with glutathione-Sepharose beads, proteins that copurified with the GST fusion proteins were detected using an anti-myc antibody (Fig. 3 A). Both mLAP16s and mLAP15s interacted with GST–LIMK1 but not with GST, indicating that the minimal region required for BMPR-II interaction with LIMK1 is contained within a 190–amino acid region located COOH terminal to the BMPR-II kinase domain.


Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1.

Foletta VC, Lim MA, Soosairajah J, Kelly AP, Stanley EG, Shannon M, He W, Das S, Massague J, Bernard O, Soosairaiah J - J. Cell Biol. (2003)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: Having established that the tail of BMPR-II was required for its interaction with LIMK1, we then sought to define the minimal region within the tail required for this interaction. The sequences responsible for the LIMK1–BMPR-II interaction were further defined by creating myc-tagged BMPR-II tail deletion constructs. Plasmids encoding truncated versions of hLAP15 (hLAP15s, amino acids 742–932) and mLAP16 (mLAP16s, amino acids 742–1038) (Fig. 1 A) were cotransfected with constructs encoding either GST–LIMK1 or GST alone. After affinity purification with glutathione-Sepharose beads, proteins that copurified with the GST fusion proteins were detected using an anti-myc antibody (Fig. 3 A). Both mLAP16s and mLAP15s interacted with GST–LIMK1 but not with GST, indicating that the minimal region required for BMPR-II interaction with LIMK1 is contained within a 190–amino acid region located COOH terminal to the BMPR-II kinase domain.

Bottom Line: Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4.A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1.This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade Parkville, Victoria 3050, Australia.

ABSTRACT
Bone morphogenetic proteins (BMPs) regulate multiple cellular processes, including cell differentiation and migration. Their signals are transduced by the kinase receptors BMPR-I and BMPR-II, leading to Smad transcription factor activation via BMPR-I. LIM kinase (LIMK) 1 is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, we isolated clones encompassing the tail region of BMPR-II. Although the BMPR-II tail is not involved in BMP signaling via Smad proteins, mutations truncating this domain are present in patients with primary pulmonary hypertension (PPH). Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4. A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1. This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.

Show MeSH
Related in: MedlinePlus