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Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.

Arya R, Kedar V, Hwang JR, McDonough H, Li HH, Taylor J, Patterson C - J. Cell Biol. (2004)

Bottom Line: Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy.MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1.MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size.

View Article: PubMed Central - PubMed

Affiliation: Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.

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MURF1 interacts with RACK1 in vivo and in vitro. (A) In a yeast two-hybrid screen the NH2 terminus of MURF1 was used as bait to screen an adult heart library. (B) To confirm the yeast two-hybrid interaction, COS 7 cells were transfected with RACK1 and/or Myc-MURF1 as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with RACK1 antibody. (C) To test the interaction between MURF1 and RACK1 in vitro, GST fusion proteins (GST alone, GST-RACK1, or GST-DB1 as a negative control) were incubated with COS 7 cell lysates that express Myc-MURF1. The protein complexes were immunoblotted with anti-Myc or anti-RACK1 antibody. (D) GST fusion proteins (GST alone or GST-MURF1) were incubated with COS 7 cell lysates expressing the indicated HA-tagged RACK1 deletions (The rapidly migrating HA-immunoreactive bands in cell lysates probably represent RACK1 degradation products). The protein complexes were immunoblotted with anti-HA, -PKCε, or -GST antibody. (E) NRVM were infected with Ad.GFP or Ad.MURF1 at the indicated multiplicities of infection (MOI), and lysates were immunoprecipitated with an antibody against PKCε. Immunoprecipitates were then blotted for the presence of RACK1 and MURF1 (using an anti-Myc antibody).
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fig1: MURF1 interacts with RACK1 in vivo and in vitro. (A) In a yeast two-hybrid screen the NH2 terminus of MURF1 was used as bait to screen an adult heart library. (B) To confirm the yeast two-hybrid interaction, COS 7 cells were transfected with RACK1 and/or Myc-MURF1 as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with RACK1 antibody. (C) To test the interaction between MURF1 and RACK1 in vitro, GST fusion proteins (GST alone, GST-RACK1, or GST-DB1 as a negative control) were incubated with COS 7 cell lysates that express Myc-MURF1. The protein complexes were immunoblotted with anti-Myc or anti-RACK1 antibody. (D) GST fusion proteins (GST alone or GST-MURF1) were incubated with COS 7 cell lysates expressing the indicated HA-tagged RACK1 deletions (The rapidly migrating HA-immunoreactive bands in cell lysates probably represent RACK1 degradation products). The protein complexes were immunoblotted with anti-HA, -PKCε, or -GST antibody. (E) NRVM were infected with Ad.GFP or Ad.MURF1 at the indicated multiplicities of infection (MOI), and lysates were immunoprecipitated with an antibody against PKCε. Immunoprecipitates were then blotted for the presence of RACK1 and MURF1 (using an anti-Myc antibody).

Mentions: To identify proteins that bind to MURF1, we screened a human heart cDNA library with a GAL4 fusion construct containing aa 1–143 of MURF1 (which contains ring finger and MURF-specific domains, but not the B-box domain known to interact with titin) using a yeast two-hybrid approach. After selection in high stringency conditions, three interacting cDNAs were isolated and sequenced. One of the cDNAs contained the COOH terminus of RACK1 (475–1144 nucleotides) in frame with GAL4. The selectivity of the MURF1–RACK1 interaction in the yeast two-hybrid reaction was confirmed by retesting in a lacZ reporter assay (Fig. 1 A).


Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.

Arya R, Kedar V, Hwang JR, McDonough H, Li HH, Taylor J, Patterson C - J. Cell Biol. (2004)

MURF1 interacts with RACK1 in vivo and in vitro. (A) In a yeast two-hybrid screen the NH2 terminus of MURF1 was used as bait to screen an adult heart library. (B) To confirm the yeast two-hybrid interaction, COS 7 cells were transfected with RACK1 and/or Myc-MURF1 as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with RACK1 antibody. (C) To test the interaction between MURF1 and RACK1 in vitro, GST fusion proteins (GST alone, GST-RACK1, or GST-DB1 as a negative control) were incubated with COS 7 cell lysates that express Myc-MURF1. The protein complexes were immunoblotted with anti-Myc or anti-RACK1 antibody. (D) GST fusion proteins (GST alone or GST-MURF1) were incubated with COS 7 cell lysates expressing the indicated HA-tagged RACK1 deletions (The rapidly migrating HA-immunoreactive bands in cell lysates probably represent RACK1 degradation products). The protein complexes were immunoblotted with anti-HA, -PKCε, or -GST antibody. (E) NRVM were infected with Ad.GFP or Ad.MURF1 at the indicated multiplicities of infection (MOI), and lysates were immunoprecipitated with an antibody against PKCε. Immunoprecipitates were then blotted for the presence of RACK1 and MURF1 (using an anti-Myc antibody).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172633&req=5

fig1: MURF1 interacts with RACK1 in vivo and in vitro. (A) In a yeast two-hybrid screen the NH2 terminus of MURF1 was used as bait to screen an adult heart library. (B) To confirm the yeast two-hybrid interaction, COS 7 cells were transfected with RACK1 and/or Myc-MURF1 as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with RACK1 antibody. (C) To test the interaction between MURF1 and RACK1 in vitro, GST fusion proteins (GST alone, GST-RACK1, or GST-DB1 as a negative control) were incubated with COS 7 cell lysates that express Myc-MURF1. The protein complexes were immunoblotted with anti-Myc or anti-RACK1 antibody. (D) GST fusion proteins (GST alone or GST-MURF1) were incubated with COS 7 cell lysates expressing the indicated HA-tagged RACK1 deletions (The rapidly migrating HA-immunoreactive bands in cell lysates probably represent RACK1 degradation products). The protein complexes were immunoblotted with anti-HA, -PKCε, or -GST antibody. (E) NRVM were infected with Ad.GFP or Ad.MURF1 at the indicated multiplicities of infection (MOI), and lysates were immunoprecipitated with an antibody against PKCε. Immunoprecipitates were then blotted for the presence of RACK1 and MURF1 (using an anti-Myc antibody).
Mentions: To identify proteins that bind to MURF1, we screened a human heart cDNA library with a GAL4 fusion construct containing aa 1–143 of MURF1 (which contains ring finger and MURF-specific domains, but not the B-box domain known to interact with titin) using a yeast two-hybrid approach. After selection in high stringency conditions, three interacting cDNAs were isolated and sequenced. One of the cDNAs contained the COOH terminus of RACK1 (475–1144 nucleotides) in frame with GAL4. The selectivity of the MURF1–RACK1 interaction in the yeast two-hybrid reaction was confirmed by retesting in a lacZ reporter assay (Fig. 1 A).

Bottom Line: Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy.MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1.MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size.

View Article: PubMed Central - PubMed

Affiliation: Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.

Show MeSH
Related in: MedlinePlus