Limits...
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 FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous HDAC5 at Ser498NRVMs were transfected with either scrambled siRNA or active siRNA duplexes targeted at FHL1 (A and B) or FHL2 (C and D) transcripts. After 48 h, cells were treated with vehicle (C) or ET1 (10 nM) (A and C) or vehicle (C) or PE (3 μM) (B and D) for 20 min. The phosphorylation status of endogenous HDAC5 was determined by immunoblot (IB) analysis using a phospho-specific pSer498 HDAC5 antibody. Protein loading was confirmed by Coomassie Blue staining. Individual immunoblots illustrate representative experiments, and histograms show quantitative data as means±S.E.M. (n=7–8). *P<0.05.
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Figure 5: Effect of FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous HDAC5 at Ser498NRVMs were transfected with either scrambled siRNA or active siRNA duplexes targeted at FHL1 (A and B) or FHL2 (C and D) transcripts. After 48 h, cells were treated with vehicle (C) or ET1 (10 nM) (A and C) or vehicle (C) or PE (3 μM) (B and D) for 20 min. The phosphorylation status of endogenous HDAC5 was determined by immunoblot (IB) analysis using a phospho-specific pSer498 HDAC5 antibody. Protein loading was confirmed by Coomassie Blue staining. Individual immunoblots illustrate representative experiments, and histograms show quantitative data as means±S.E.M. (n=7–8). *P<0.05.

Mentions: To explore whether the attenuation of PKD activation is paralleled by reduced phosphorylation of a functionally important cellular PKD substrate, we also studied the effects of FHL1 and FHL2 knockdown on the phosphorylation status of HDAC5 at Ser498, an established PKD substrate in cardiac myocytes [10,11]. Selective FHL isoform knockdown in NRVMs attenuated HDAC5 phosphorylation in response to stimulation with ET1 or PE (Figure 5), in a manner that mirrored the effects of these loss-of-function interventions on PKD phosphorylation (Figure 4). Thus selective knockdown of FHL1 expression significantly attenuated the increase in HDAC5 phosphorylation in response to ET1 compared with control cells transfected with a scrambled siRNA sequence (Figure 5A), but had a non-significant effect on the increase in HDAC5 phosphorylation in response to PE (Figure 5B). In contrast, selective knockdown of FHL2 expression significantly attenuated the increases in HDAC5 phosphorylation in response to both ET1 (Figure 5C) and PE (Figure 5D). Therefore it appears that, in response to neurohormonal stimulation in NRVMs, FHL1 and FHL2 facilitate not only PKD activation, but also phosphorylation of the downstream PKD substrate HDAC5. Once again, the effects of simultaneous knockdown of both FHL1 and FHL2 on HDAC5 phosphorylation were similar to the effects of selective FHL2 knockdown, with no indication of an additive effect (Supplementary Figure S2).


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 FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous HDAC5 at Ser498NRVMs were transfected with either scrambled siRNA or active siRNA duplexes targeted at FHL1 (A and B) or FHL2 (C and D) transcripts. After 48 h, cells were treated with vehicle (C) or ET1 (10 nM) (A and C) or vehicle (C) or PE (3 μM) (B and D) for 20 min. The phosphorylation status of endogenous HDAC5 was determined by immunoblot (IB) analysis using a phospho-specific pSer498 HDAC5 antibody. Protein loading was confirmed by Coomassie Blue staining. Individual immunoblots illustrate representative experiments, and histograms show quantitative data as means±S.E.M. (n=7–8). *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Effect of FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous HDAC5 at Ser498NRVMs were transfected with either scrambled siRNA or active siRNA duplexes targeted at FHL1 (A and B) or FHL2 (C and D) transcripts. After 48 h, cells were treated with vehicle (C) or ET1 (10 nM) (A and C) or vehicle (C) or PE (3 μM) (B and D) for 20 min. The phosphorylation status of endogenous HDAC5 was determined by immunoblot (IB) analysis using a phospho-specific pSer498 HDAC5 antibody. Protein loading was confirmed by Coomassie Blue staining. Individual immunoblots illustrate representative experiments, and histograms show quantitative data as means±S.E.M. (n=7–8). *P<0.05.
Mentions: To explore whether the attenuation of PKD activation is paralleled by reduced phosphorylation of a functionally important cellular PKD substrate, we also studied the effects of FHL1 and FHL2 knockdown on the phosphorylation status of HDAC5 at Ser498, an established PKD substrate in cardiac myocytes [10,11]. Selective FHL isoform knockdown in NRVMs attenuated HDAC5 phosphorylation in response to stimulation with ET1 or PE (Figure 5), in a manner that mirrored the effects of these loss-of-function interventions on PKD phosphorylation (Figure 4). Thus selective knockdown of FHL1 expression significantly attenuated the increase in HDAC5 phosphorylation in response to ET1 compared with control cells transfected with a scrambled siRNA sequence (Figure 5A), but had a non-significant effect on the increase in HDAC5 phosphorylation in response to PE (Figure 5B). In contrast, selective knockdown of FHL2 expression significantly attenuated the increases in HDAC5 phosphorylation in response to both ET1 (Figure 5C) and PE (Figure 5D). Therefore it appears that, in response to neurohormonal stimulation in NRVMs, FHL1 and FHL2 facilitate not only PKD activation, but also phosphorylation of the downstream PKD substrate HDAC5. Once again, the effects of simultaneous knockdown of both FHL1 and FHL2 on HDAC5 phosphorylation were similar to the effects of selective FHL2 knockdown, with no indication of an additive effect (Supplementary Figure S2).

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