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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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Effects of MURF1 on components of the FAK signaling pathway. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE or PMA. NRVM lysates were separated by SDS-PAGE. Blots were incubated with anti-pFAK (397Y) antibody and anti-FAK antibody (loading control). (B) NRVM cell lysates treated as in A were immunoprecipitated with anti-paxillin antibody followed by immunoblotting with anti-phosphotyrosine (PY) antibody. (C) To test for activation of MAP kinase signaling, NRVM cell lysates were analyzed by Western blotting with anti-pERK antibody and anti-ERK antibody (loading control). The cumulative results are presented as fold increase in ERK activity from basal level ± SEM.
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fig7: Effects of MURF1 on components of the FAK signaling pathway. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE or PMA. NRVM lysates were separated by SDS-PAGE. Blots were incubated with anti-pFAK (397Y) antibody and anti-FAK antibody (loading control). (B) NRVM cell lysates treated as in A were immunoprecipitated with anti-paxillin antibody followed by immunoblotting with anti-phosphotyrosine (PY) antibody. (C) To test for activation of MAP kinase signaling, NRVM cell lysates were analyzed by Western blotting with anti-pERK antibody and anti-ERK antibody (loading control). The cumulative results are presented as fold increase in ERK activity from basal level ± SEM.

Mentions: Tyrosine phosphorylation is a major covalent modification driving protein–protein interactions required for focal adhesion assembly. To determine the kinetics and extent of tyrosine phosphorylation of focal adhesion–associated proteins, FAK was immunoblotted with a phospho-FAK antibody that specifically recognizes the phosphorylation of tyrosine 397 (Schaller et al., 1994), which is crucial for FAK activation. An increase in tyrosine phosphorylation of FAK was observed after 15–20 min of PE treatment in control cells (Fig. 7 A). This phosphorylation of tyrosine 397 was blocked by increasing levels of MURF1. In addition, we immunoprecipitated paxillin from PE-treated cells and tested its phosphorylation status by Western blotting with anti-phosphotyrosine antibody. As expected, paxillin phosphorylation occurred rapidly after PE or PMA treatment, and MuRF1 inhibited the phosphorylation of paxillin by PE or PMA (Fig. 7 B). Previous papers have shown that tyrosine phosphorylation and activation of FAK can activate ERK1/2 signaling cascades (Govindarajan et al., 2000), and that ERK2 is the downstream target of FAK that is activated during assembly of focal adhesion proteins in cardiac myocytes (Taylor et al., 2000). Similar to the inhibition of FAK phosphorylation, we observed inhibition of ERK2 phosphorylation in Ad.MURF1-infected cardiomyocytes after 15–20 min of PE or PMA treatment (Fig. 7 C). Together, these observations indicate that the ability of MURF1 to inhibit activation of PKCε leads to impaired focal adhesion assembly and arrests signaling downstream of FAK in PE- or PMA-stimulated NRVM.


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)

Effects of MURF1 on components of the FAK signaling pathway. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE or PMA. NRVM lysates were separated by SDS-PAGE. Blots were incubated with anti-pFAK (397Y) antibody and anti-FAK antibody (loading control). (B) NRVM cell lysates treated as in A were immunoprecipitated with anti-paxillin antibody followed by immunoblotting with anti-phosphotyrosine (PY) antibody. (C) To test for activation of MAP kinase signaling, NRVM cell lysates were analyzed by Western blotting with anti-pERK antibody and anti-ERK antibody (loading control). The cumulative results are presented as fold increase in ERK activity from basal level ± SEM.
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Related In: Results  -  Collection

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fig7: Effects of MURF1 on components of the FAK signaling pathway. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE or PMA. NRVM lysates were separated by SDS-PAGE. Blots were incubated with anti-pFAK (397Y) antibody and anti-FAK antibody (loading control). (B) NRVM cell lysates treated as in A were immunoprecipitated with anti-paxillin antibody followed by immunoblotting with anti-phosphotyrosine (PY) antibody. (C) To test for activation of MAP kinase signaling, NRVM cell lysates were analyzed by Western blotting with anti-pERK antibody and anti-ERK antibody (loading control). The cumulative results are presented as fold increase in ERK activity from basal level ± SEM.
Mentions: Tyrosine phosphorylation is a major covalent modification driving protein–protein interactions required for focal adhesion assembly. To determine the kinetics and extent of tyrosine phosphorylation of focal adhesion–associated proteins, FAK was immunoblotted with a phospho-FAK antibody that specifically recognizes the phosphorylation of tyrosine 397 (Schaller et al., 1994), which is crucial for FAK activation. An increase in tyrosine phosphorylation of FAK was observed after 15–20 min of PE treatment in control cells (Fig. 7 A). This phosphorylation of tyrosine 397 was blocked by increasing levels of MURF1. In addition, we immunoprecipitated paxillin from PE-treated cells and tested its phosphorylation status by Western blotting with anti-phosphotyrosine antibody. As expected, paxillin phosphorylation occurred rapidly after PE or PMA treatment, and MuRF1 inhibited the phosphorylation of paxillin by PE or PMA (Fig. 7 B). Previous papers have shown that tyrosine phosphorylation and activation of FAK can activate ERK1/2 signaling cascades (Govindarajan et al., 2000), and that ERK2 is the downstream target of FAK that is activated during assembly of focal adhesion proteins in cardiac myocytes (Taylor et al., 2000). Similar to the inhibition of FAK phosphorylation, we observed inhibition of ERK2 phosphorylation in Ad.MURF1-infected cardiomyocytes after 15–20 min of PE or PMA treatment (Fig. 7 C). Together, these observations indicate that the ability of MURF1 to inhibit activation of PKCε leads to impaired focal adhesion assembly and arrests signaling downstream of FAK in PE- or PMA-stimulated NRVM.

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