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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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).
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fig5: 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).

Mentions: To demonstrate that the interaction between LIMK1 and BMPR-II has biological significance, we studied the effect of BMP4 on the activity of LIMK1. As the only known substrate of LIMK1 is cofilin, change in the level of phospho-cofilin is a surrogate measure of LIMK1 activity. COS-7 cells were serum starved and then incubated in the presence or absence of 10 ng/ml BMP4 for periods of 5–30 min and compared with unstimulated cells. Cell lysates were subjected to immunoblotting with anti–phospho-cofilin antibodies. Western blot filters were subsequently stripped and reprobed with anti-cofilin antibodies to allow the absolute amount of cofilin to be measured. Incubation with BMP4 for 5 min resulted in a sevenfold increase in the level of phospho-cofilin compared with controls. An increased level of phospho-cofilin was maintained, but to a lesser extent after 10 and 30 min incubation with BMP4, relative to that found in untreated cells (Fig. 5 A).


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)

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).
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fig5: 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).
Mentions: To demonstrate that the interaction between LIMK1 and BMPR-II has biological significance, we studied the effect of BMP4 on the activity of LIMK1. As the only known substrate of LIMK1 is cofilin, change in the level of phospho-cofilin is a surrogate measure of LIMK1 activity. COS-7 cells were serum starved and then incubated in the presence or absence of 10 ng/ml BMP4 for periods of 5–30 min and compared with unstimulated cells. Cell lysates were subjected to immunoblotting with anti–phospho-cofilin antibodies. Western blot filters were subsequently stripped and reprobed with anti-cofilin antibodies to allow the absolute amount of cofilin to be measured. Incubation with BMP4 for 5 min resulted in a sevenfold increase in the level of phospho-cofilin compared with controls. An increased level of phospho-cofilin was maintained, but to a lesser extent after 10 and 30 min incubation with BMP4, relative to that found in untreated cells (Fig. 5 A).

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