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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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).
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fig1: 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).

Mentions: To identify new molecules that regulate LIMK1 activity, we conducted yeast two-hybrid screens (Fields and Song, 1989) to isolate LIMK1-associated proteins (LAPs). After screening of mouse embryonic and human brain cDNA libraries using full-length LIMK1 fused to the GAL4 DNA binding domain as bait, two clones from each library interacted strongly with LIMK1 but did not interact with two negative control baits (Jun and Lck) or empty vector. These four clones were picked for further analysis as they demonstrated the greatest β-galactosidase activity and hence the strength of interaction in this particular assay system. Sequence analysis of these LAPs (mLAP16, mLAP22, hLAP15, and hLAP41) revealed that they all contained cDNA inserts corresponding to a region within the cytoplasmic tail of BMPR-II (Fig. 1 A). The interaction between LIMK1 and the tail of BMPR-II was confirmed in mammalian cells. The cDNAs of the three LAPs (mLAP16, hLAP15, and hLAP41) were subcloned into GFP-encoding expression vectors and were coexpressed with either FLAG-tagged LIMK1 or Btk, a cytoplasmic protein kinase unrelated in function to LIMK1, in COS-7 cells. The FLAG-tagged proteins were immunopurified with FLAG M2 beads, and interacting proteins were detected by Western blotting with an anti-GFP antibody (Fig. 1 B). All three LAPs interacted with LIMK1 (lanes 7–9) but not with Btk (lanes 4–6) or with the FLAG beads alone (lanes 1–3).


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)

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172847&req=5

fig1: 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).
Mentions: To identify new molecules that regulate LIMK1 activity, we conducted yeast two-hybrid screens (Fields and Song, 1989) to isolate LIMK1-associated proteins (LAPs). After screening of mouse embryonic and human brain cDNA libraries using full-length LIMK1 fused to the GAL4 DNA binding domain as bait, two clones from each library interacted strongly with LIMK1 but did not interact with two negative control baits (Jun and Lck) or empty vector. These four clones were picked for further analysis as they demonstrated the greatest β-galactosidase activity and hence the strength of interaction in this particular assay system. Sequence analysis of these LAPs (mLAP16, mLAP22, hLAP15, and hLAP41) revealed that they all contained cDNA inserts corresponding to a region within the cytoplasmic tail of BMPR-II (Fig. 1 A). The interaction between LIMK1 and the tail of BMPR-II was confirmed in mammalian cells. The cDNAs of the three LAPs (mLAP16, hLAP15, and hLAP41) were subcloned into GFP-encoding expression vectors and were coexpressed with either FLAG-tagged LIMK1 or Btk, a cytoplasmic protein kinase unrelated in function to LIMK1, in COS-7 cells. The FLAG-tagged proteins were immunopurified with FLAG M2 beads, and interacting proteins were detected by Western blotting with an anti-GFP antibody (Fig. 1 B). All three LAPs interacted with LIMK1 (lanes 7–9) but not with Btk (lanes 4–6) or with the FLAG beads alone (lanes 1–3).

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