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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172847&req=5

fig2: 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.

Mentions: It is possible that the interaction between overexpressed BMPR-II and GST–LIMK1 was driven by the large amounts of the proteins produced in the COS cell overexpression system. To address this issue, we sought to determine if the interaction occurred between endogenously expressed proteins in tissue culture cells. Initially, we performed a survey of human and mouse cell lines for expression of LIMK1, BMPR-II, and the alternative spliced protein lacking the cytoplasmic tail, BMPR-IIs (Liu et al., 1995). RNAs encoding the BMPR-IIs were of particular interest because our results implied that cells might be able to modulate the amount of BMPR-II–LIMK1 complex by altering the proportion of BMPR-II to BMPR-IIs. However, Northern blot analysis of most of the cell lines examined, which included human HaCaT keratinocytes, HPL1 lung epithelial cells, HUVEC umbilical cord endothelial cells, WI38 lung fibroblasts, A549 lung carcinoma, HepG2 hepatoma, and mouse NMuMG mammary epithelial cells, C2C12 myoblasts, and NIH3T3 mouse embryo fibroblasts, showed mRNA expression of BMPRII and Limk1 but not BMPRIIs (unpublished data). To facilitate the analysis of BMPR-II–LIMK1 interactions, we raised rabbit polyclonal antibodies against the kinase domain and the cytoplasmic tail of BMPR-II domain (Fig. 2 A) in addition to using a mouse monoclonal anti-LIMK1 antibody that recognizes endogenous LIMK1 protein. Western immunoblotting of cell lysates using these antibodies after a survey of selected cell lines revealed that BMPR-II is expressed in all cell lines tested (Fig. 2 B; unpublished data). NIH3T3 fibroblasts, which express an intermediate level of BMPR-II, also expressed a moderate level of LIMK1 (Fig. 2 C) and were used to investigate the interaction between these two endogenous proteins. Immunoprecipitation of cell lysates with anti–BMPR-II (tail) antibodies followed by Western immunoblotting of these precipitates with mouse anti-LIMK1 monoclonal antibody demonstrated an interaction between endogenous BMPR-II and LIMK1 proteins (Fig. 2 C).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: It is possible that the interaction between overexpressed BMPR-II and GST–LIMK1 was driven by the large amounts of the proteins produced in the COS cell overexpression system. To address this issue, we sought to determine if the interaction occurred between endogenously expressed proteins in tissue culture cells. Initially, we performed a survey of human and mouse cell lines for expression of LIMK1, BMPR-II, and the alternative spliced protein lacking the cytoplasmic tail, BMPR-IIs (Liu et al., 1995). RNAs encoding the BMPR-IIs were of particular interest because our results implied that cells might be able to modulate the amount of BMPR-II–LIMK1 complex by altering the proportion of BMPR-II to BMPR-IIs. However, Northern blot analysis of most of the cell lines examined, which included human HaCaT keratinocytes, HPL1 lung epithelial cells, HUVEC umbilical cord endothelial cells, WI38 lung fibroblasts, A549 lung carcinoma, HepG2 hepatoma, and mouse NMuMG mammary epithelial cells, C2C12 myoblasts, and NIH3T3 mouse embryo fibroblasts, showed mRNA expression of BMPRII and Limk1 but not BMPRIIs (unpublished data). To facilitate the analysis of BMPR-II–LIMK1 interactions, we raised rabbit polyclonal antibodies against the kinase domain and the cytoplasmic tail of BMPR-II domain (Fig. 2 A) in addition to using a mouse monoclonal anti-LIMK1 antibody that recognizes endogenous LIMK1 protein. Western immunoblotting of cell lysates using these antibodies after a survey of selected cell lines revealed that BMPR-II is expressed in all cell lines tested (Fig. 2 B; unpublished data). NIH3T3 fibroblasts, which express an intermediate level of BMPR-II, also expressed a moderate level of LIMK1 (Fig. 2 C) and were used to investigate the interaction between these two endogenous proteins. Immunoprecipitation of cell lysates with anti–BMPR-II (tail) antibodies followed by Western immunoblotting of these precipitates with mouse anti-LIMK1 monoclonal antibody demonstrated an interaction between endogenous BMPR-II and LIMK1 proteins (Fig. 2 C).

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