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PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Lee WR, Sacharidou A, Behling-Kelly E, Oltmann SC, Zhu W, Ahmed M, Gerard RD, Hui DY, Abe J, Shaul PW, Mineo C - PLoS ONE (2015)

Bottom Line: Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM.PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion.Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF.

View Article: PubMed Central - PubMed

Affiliation: Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.

No MeSH data available.


Related in: MedlinePlus

Bcr is expressed in VSM cells and interacts with PDZK1.A. PDZK1+/+ or PDZK1-/- VSM cells were transfected with control dsRNA (Con) or dsRNA targeting Bcr, and Bcr expression was evaluated by immunoblot analysis 24h later. B. Wild-type VSM cells were infected with adenovirus expressing protein G sequence-containing Tag alone (lane 1) or Tag-PDZK1 (lane 2), and 60h later the cells were lysed and Tag or Tag-PDZK1 were precipitated using IgG sepharose. Tag and Tag-PDZK1 were detected by HRP-linked secondary antibody binding to the Protein G sequence in the Tag (left panel). The pull-down of Bcr was detected by immunoblotting of samples from Tag-PDZK1 infected cells (right panel). C. Endogenous Bcr was immunoprecipitated from PDZK1+/+ or PDZK-/- VSM cells by anti-Bcr antibody, and the presence of Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting. D. Endogenous PDZK1 was immunoprecipitated by anti-PDZK1 antibody, and Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting.
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pone.0124494.g004: Bcr is expressed in VSM cells and interacts with PDZK1.A. PDZK1+/+ or PDZK1-/- VSM cells were transfected with control dsRNA (Con) or dsRNA targeting Bcr, and Bcr expression was evaluated by immunoblot analysis 24h later. B. Wild-type VSM cells were infected with adenovirus expressing protein G sequence-containing Tag alone (lane 1) or Tag-PDZK1 (lane 2), and 60h later the cells were lysed and Tag or Tag-PDZK1 were precipitated using IgG sepharose. Tag and Tag-PDZK1 were detected by HRP-linked secondary antibody binding to the Protein G sequence in the Tag (left panel). The pull-down of Bcr was detected by immunoblotting of samples from Tag-PDZK1 infected cells (right panel). C. Endogenous Bcr was immunoprecipitated from PDZK1+/+ or PDZK-/- VSM cells by anti-Bcr antibody, and the presence of Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting. D. Endogenous PDZK1 was immunoprecipitated by anti-PDZK1 antibody, and Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting.

Mentions: Bcr was originally identified as the breakpoint of the Philadelphia chromosome translocation associated with chronic myelogenous leukemia [27,28]. It is a 160 kDa protein that contains a number of putative functional domains, including a C-terminal sequence (STEV) that is a ligand for PDZ domains [27,29,30]. Previous studies demonstrated that Bcr is expressed in VSM cells, and that its function enhances VSM proliferation [31,32]. The expression of Bcr in primary VSM cells was confirmed by immunoblot analysis of cells transfected with control siRNA versus siRNA targeting the kinase (Fig 4A), and there was no difference in the levels of Bcr expression in PDZK1+/+ versus PDZK1-/- VSM. The interaction of Bcr with PDZK1 in VSM cells was evaluated by pull-down using protein G in the TAP-Tag (Fig 4B). VSM cells from wild-type mice were infected with adenovirus encoding either the TAP-Tag alone, which contains two protein G sequences, or the Tag-PDZK1. Tag or Tag-PDZK1 proteins were captured using IgG Sepharose beads, and Bcr co-precipitated with PDZK1 was detected by immunoblot analysis. Interaction between endogenous PDZK1 and Bcr was evaluated in VSM cells from PDKZ1+/+ or PDZK1-/- mice in coimmunoprecipitation experiments. In PDZK1+/+ cells, PDZK1 was coimmunoprecipitated with Bcr (Fig 4C), and in a reciprocal manner, Bcr was coimmunoprecipitated with PDZK1 (Fig 4D). These results indicate that Bcr is a PDZK1 interacting protein in VSM cells.


PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Lee WR, Sacharidou A, Behling-Kelly E, Oltmann SC, Zhu W, Ahmed M, Gerard RD, Hui DY, Abe J, Shaul PW, Mineo C - PLoS ONE (2015)

Bcr is expressed in VSM cells and interacts with PDZK1.A. PDZK1+/+ or PDZK1-/- VSM cells were transfected with control dsRNA (Con) or dsRNA targeting Bcr, and Bcr expression was evaluated by immunoblot analysis 24h later. B. Wild-type VSM cells were infected with adenovirus expressing protein G sequence-containing Tag alone (lane 1) or Tag-PDZK1 (lane 2), and 60h later the cells were lysed and Tag or Tag-PDZK1 were precipitated using IgG sepharose. Tag and Tag-PDZK1 were detected by HRP-linked secondary antibody binding to the Protein G sequence in the Tag (left panel). The pull-down of Bcr was detected by immunoblotting of samples from Tag-PDZK1 infected cells (right panel). C. Endogenous Bcr was immunoprecipitated from PDZK1+/+ or PDZK-/- VSM cells by anti-Bcr antibody, and the presence of Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting. D. Endogenous PDZK1 was immunoprecipitated by anti-PDZK1 antibody, and Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting.
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Related In: Results  -  Collection

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pone.0124494.g004: Bcr is expressed in VSM cells and interacts with PDZK1.A. PDZK1+/+ or PDZK1-/- VSM cells were transfected with control dsRNA (Con) or dsRNA targeting Bcr, and Bcr expression was evaluated by immunoblot analysis 24h later. B. Wild-type VSM cells were infected with adenovirus expressing protein G sequence-containing Tag alone (lane 1) or Tag-PDZK1 (lane 2), and 60h later the cells were lysed and Tag or Tag-PDZK1 were precipitated using IgG sepharose. Tag and Tag-PDZK1 were detected by HRP-linked secondary antibody binding to the Protein G sequence in the Tag (left panel). The pull-down of Bcr was detected by immunoblotting of samples from Tag-PDZK1 infected cells (right panel). C. Endogenous Bcr was immunoprecipitated from PDZK1+/+ or PDZK-/- VSM cells by anti-Bcr antibody, and the presence of Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting. D. Endogenous PDZK1 was immunoprecipitated by anti-PDZK1 antibody, and Bcr and PDZK1 in the immunoprecipitates was detected by immunoblotting.
Mentions: Bcr was originally identified as the breakpoint of the Philadelphia chromosome translocation associated with chronic myelogenous leukemia [27,28]. It is a 160 kDa protein that contains a number of putative functional domains, including a C-terminal sequence (STEV) that is a ligand for PDZ domains [27,29,30]. Previous studies demonstrated that Bcr is expressed in VSM cells, and that its function enhances VSM proliferation [31,32]. The expression of Bcr in primary VSM cells was confirmed by immunoblot analysis of cells transfected with control siRNA versus siRNA targeting the kinase (Fig 4A), and there was no difference in the levels of Bcr expression in PDZK1+/+ versus PDZK1-/- VSM. The interaction of Bcr with PDZK1 in VSM cells was evaluated by pull-down using protein G in the TAP-Tag (Fig 4B). VSM cells from wild-type mice were infected with adenovirus encoding either the TAP-Tag alone, which contains two protein G sequences, or the Tag-PDZK1. Tag or Tag-PDZK1 proteins were captured using IgG Sepharose beads, and Bcr co-precipitated with PDZK1 was detected by immunoblot analysis. Interaction between endogenous PDZK1 and Bcr was evaluated in VSM cells from PDKZ1+/+ or PDZK1-/- mice in coimmunoprecipitation experiments. In PDZK1+/+ cells, PDZK1 was coimmunoprecipitated with Bcr (Fig 4C), and in a reciprocal manner, Bcr was coimmunoprecipitated with PDZK1 (Fig 4D). These results indicate that Bcr is a PDZK1 interacting protein in VSM cells.

Bottom Line: Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM.PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion.Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF.

View Article: PubMed Central - PubMed

Affiliation: Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.

No MeSH data available.


Related in: MedlinePlus