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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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.
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fig4: 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.

Mentions: As endogenous LIMK1 and BMPR-II were found to associate, we examined if this interaction affected their known activities. To assess this, the ability of LIMK1 to phosphorylate cofilin using in vitro kinase assays in the presence or absence of BMPR-II was examined. Myc-tagged LIMK1 and myc-tagged BMPR-II were expressed separately in COS cells and then immunoprecipitated with anti-myc antibodies either alone or together after the lysates were combined. The immunocomplexes were labeled in vitro with [32P]γ-ATP to determine their level of activity. Both kinase molecules exhibited high levels of autophosphorylation when assayed individually (Fig. 4 A). In addition, LIMK1, but not BMPR-II, was able to phosphorylate 5 μg of recombinant GST–cofilin present in all samples (Fig. 4 A). However, when LIMK1 and BMPR-II were coimmunopurified, a decrease in the level of phosphorylated GST–cofilin was observed with no change evident in the level of phosphorylation of LIMK1 or BMPR-II. All samples were normalized for the amount of LIMK1 protein present, as determined by densitometry analysis of immunoblotted LIMK1. The level of GST–cofilin phosphorylation by LIMK1 was reduced up to fivefold after LIMK1 interaction with BMPR-II in six individual experiments performed where an average of 0.45 ± 0.10 SEM was measured and compared with 1.0, the value given to the level of phosphorylated GST–cofilin in the presence of myc-tagged LIMK1 alone (Fig. 4 A). A reduction in GST–cofilin phosphorylation was confirmed in experiments where protein lysates containing FLAG-tagged or GFP-tagged LIMK1 proteins were combined with myc-tagged BMPR-II protein lysates and coimmunopurified using either anti-FLAG or anti-GFP antibodies, respectively (Fig. 4 B), and tested for their ability to phosphorylate cofilin. After normalization for LIMK1 protein, a reproducible decrease in the phosphorylation of GST–cofilin was evident (Fig. 4 B), as shown in Fig. 4 A. In addition, the activity of the LIMK1 kinase domain alone, as judged by its ability to phosphorylate cofilin, was not affected by the presence of BMPR-II (Fig. 4 B), indicating that the interaction between the two molecules leads to down-regulation of LIMK1 activity. Moreover, the presence of a tailless BMPR-II receptor did not affect the activity of LIMK1 (Fig. 4 C). The PPH-like mutant BMPR-II (R873X) did not affect the ability of LIMK1 to phosphorylate cofilin (unpublished data), supporting the notion that PPH mutations may compromise the regulation of LIMK1. Finally, in a separate set of experiments, the interaction of LIMK1 with BMPR-II did not appear to alter the ability of BMPR-II to phosphorylate BMPR-IA (unpublished data).


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)

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.
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Related In: Results  -  Collection

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fig4: 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.
Mentions: As endogenous LIMK1 and BMPR-II were found to associate, we examined if this interaction affected their known activities. To assess this, the ability of LIMK1 to phosphorylate cofilin using in vitro kinase assays in the presence or absence of BMPR-II was examined. Myc-tagged LIMK1 and myc-tagged BMPR-II were expressed separately in COS cells and then immunoprecipitated with anti-myc antibodies either alone or together after the lysates were combined. The immunocomplexes were labeled in vitro with [32P]γ-ATP to determine their level of activity. Both kinase molecules exhibited high levels of autophosphorylation when assayed individually (Fig. 4 A). In addition, LIMK1, but not BMPR-II, was able to phosphorylate 5 μg of recombinant GST–cofilin present in all samples (Fig. 4 A). However, when LIMK1 and BMPR-II were coimmunopurified, a decrease in the level of phosphorylated GST–cofilin was observed with no change evident in the level of phosphorylation of LIMK1 or BMPR-II. All samples were normalized for the amount of LIMK1 protein present, as determined by densitometry analysis of immunoblotted LIMK1. The level of GST–cofilin phosphorylation by LIMK1 was reduced up to fivefold after LIMK1 interaction with BMPR-II in six individual experiments performed where an average of 0.45 ± 0.10 SEM was measured and compared with 1.0, the value given to the level of phosphorylated GST–cofilin in the presence of myc-tagged LIMK1 alone (Fig. 4 A). A reduction in GST–cofilin phosphorylation was confirmed in experiments where protein lysates containing FLAG-tagged or GFP-tagged LIMK1 proteins were combined with myc-tagged BMPR-II protein lysates and coimmunopurified using either anti-FLAG or anti-GFP antibodies, respectively (Fig. 4 B), and tested for their ability to phosphorylate cofilin. After normalization for LIMK1 protein, a reproducible decrease in the phosphorylation of GST–cofilin was evident (Fig. 4 B), as shown in Fig. 4 A. In addition, the activity of the LIMK1 kinase domain alone, as judged by its ability to phosphorylate cofilin, was not affected by the presence of BMPR-II (Fig. 4 B), indicating that the interaction between the two molecules leads to down-regulation of LIMK1 activity. Moreover, the presence of a tailless BMPR-II receptor did not affect the activity of LIMK1 (Fig. 4 C). The PPH-like mutant BMPR-II (R873X) did not affect the ability of LIMK1 to phosphorylate cofilin (unpublished data), supporting the notion that PPH mutations may compromise the regulation of LIMK1. Finally, in a separate set of experiments, the interaction of LIMK1 with BMPR-II did not appear to alter the ability of BMPR-II to phosphorylate BMPR-IA (unpublished data).

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