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Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes.

Stathopoulou K, Cuello F, Candasamy AJ, Kemp EM, Ehler E, Haworth RS, Avkiran M - Biochem. J. (2014)

Bottom Line: In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli.Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine.We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

View Article: PubMed Central - PubMed

Affiliation: *Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.

ABSTRACT
PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α₁-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

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Effect of pharmacological PKD inhibition on ET1- and PE-induced HDAC5 phosphorylation and MEF2 activation(A) NRVMs were treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 20 min, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). PKD autophosphorylation at Ser916, total PKD expression and HDAC5 phosphorylation at Ser498 were assessed by immunoblot (IB) analysis using appropriate antibodies, as indicated. Membranes were stained with Coomassie Blue to confirm equal protein loading. Data are representative of three independent experiments. (B and C) NRVMs were co-transfected with a 3×MEF2-firefly luciferase reporter vector and a Renilla luciferase control vector and treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 18–24 h, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). Luciferase activity in cell lysates was assessed by in vitro luminescence assays, with luciferase activity normalized for Renilla luciferase activity in each sample to correct for transfection efficiency. Histograms show quantitative data as means±S.E.M. (n=4). *P<0.05.
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Figure 7: Effect of pharmacological PKD inhibition on ET1- and PE-induced HDAC5 phosphorylation and MEF2 activation(A) NRVMs were treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 20 min, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). PKD autophosphorylation at Ser916, total PKD expression and HDAC5 phosphorylation at Ser498 were assessed by immunoblot (IB) analysis using appropriate antibodies, as indicated. Membranes were stained with Coomassie Blue to confirm equal protein loading. Data are representative of three independent experiments. (B and C) NRVMs were co-transfected with a 3×MEF2-firefly luciferase reporter vector and a Renilla luciferase control vector and treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 18–24 h, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). Luciferase activity in cell lysates was assessed by in vitro luminescence assays, with luciferase activity normalized for Renilla luciferase activity in each sample to correct for transfection efficiency. Histograms show quantitative data as means±S.E.M. (n=4). *P<0.05.

Mentions: One possible explanation for this lack of effect of FHL isoform knockdown on MEF2 activation, despite a significant attenuation of PKD activation, is that under our experimental conditions PKD activity is not an important mediator of MEF2 activation in response to ET1 or PE. To help confirm the role of PKD activity in MEF2 activation in NRVMs, therefore, we also investigated the effects of the selective PKD inhibitor BPKDi [20,23] under the same experimental conditions. Consistent with previous work [23], pre-treatment of NRVMs with BPKDi completely abolished ET1- and PE-induced increases in both PKD autophosphorylation and HDAC5 phosphorylation (Figure 7A). Furthermore, pre-treatment with BPKDi significantly attenuated ET1- and PE-induced increases in MEF2 activity (Figures 7B and 7C). Thus it appears that FHL1 and FHL2 facilitate PKD activation and HDAC5 phosphorylation, but not MEF2 activation following ET1 and PE stimulation, despite the fact that PKD activity is indeed an important mediator of both HDAC5 phosphorylation and MEF2 activation under these experimental conditions.


Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes.

Stathopoulou K, Cuello F, Candasamy AJ, Kemp EM, Ehler E, Haworth RS, Avkiran M - Biochem. J. (2014)

Effect of pharmacological PKD inhibition on ET1- and PE-induced HDAC5 phosphorylation and MEF2 activation(A) NRVMs were treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 20 min, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). PKD autophosphorylation at Ser916, total PKD expression and HDAC5 phosphorylation at Ser498 were assessed by immunoblot (IB) analysis using appropriate antibodies, as indicated. Membranes were stained with Coomassie Blue to confirm equal protein loading. Data are representative of three independent experiments. (B and C) NRVMs were co-transfected with a 3×MEF2-firefly luciferase reporter vector and a Renilla luciferase control vector and treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 18–24 h, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). Luciferase activity in cell lysates was assessed by in vitro luminescence assays, with luciferase activity normalized for Renilla luciferase activity in each sample to correct for transfection efficiency. Histograms show quantitative data as means±S.E.M. (n=4). *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Effect of pharmacological PKD inhibition on ET1- and PE-induced HDAC5 phosphorylation and MEF2 activation(A) NRVMs were treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 20 min, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). PKD autophosphorylation at Ser916, total PKD expression and HDAC5 phosphorylation at Ser498 were assessed by immunoblot (IB) analysis using appropriate antibodies, as indicated. Membranes were stained with Coomassie Blue to confirm equal protein loading. Data are representative of three independent experiments. (B and C) NRVMs were co-transfected with a 3×MEF2-firefly luciferase reporter vector and a Renilla luciferase control vector and treated with vehicle (C), ET1 (10 nM) or PE (3 μM) for 18–24 h, following a 30-min pre-treatment with vehicle or the BPKDi (3 μM). Luciferase activity in cell lysates was assessed by in vitro luminescence assays, with luciferase activity normalized for Renilla luciferase activity in each sample to correct for transfection efficiency. Histograms show quantitative data as means±S.E.M. (n=4). *P<0.05.
Mentions: One possible explanation for this lack of effect of FHL isoform knockdown on MEF2 activation, despite a significant attenuation of PKD activation, is that under our experimental conditions PKD activity is not an important mediator of MEF2 activation in response to ET1 or PE. To help confirm the role of PKD activity in MEF2 activation in NRVMs, therefore, we also investigated the effects of the selective PKD inhibitor BPKDi [20,23] under the same experimental conditions. Consistent with previous work [23], pre-treatment of NRVMs with BPKDi completely abolished ET1- and PE-induced increases in both PKD autophosphorylation and HDAC5 phosphorylation (Figure 7A). Furthermore, pre-treatment with BPKDi significantly attenuated ET1- and PE-induced increases in MEF2 activity (Figures 7B and 7C). Thus it appears that FHL1 and FHL2 facilitate PKD activation and HDAC5 phosphorylation, but not MEF2 activation following ET1 and PE stimulation, despite the fact that PKD activity is indeed an important mediator of both HDAC5 phosphorylation and MEF2 activation under these experimental conditions.

Bottom Line: In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli.Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine.We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

View Article: PubMed Central - PubMed

Affiliation: *Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.

ABSTRACT
PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α₁-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

Show MeSH
Related in: MedlinePlus