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.

Show MeSH

Related in: MedlinePlus

Effect of FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous PKDNRVMs 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 μ M) (B and D) for 20 min. Phosphorylation status of endogenous PKD at Ser916 and Ser744/Ser748 was determined by immunoblot (IB) analysis using appropriate phospho-specific antibodies. Protein loading was confirmed using an anti-PKD antibody and 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

License
getmorefigures.php?uid=PMC3927927&req=5

Figure 4: Effect of FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous PKDNRVMs 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 μ M) (B and D) for 20 min. Phosphorylation status of endogenous PKD at Ser916 and Ser744/Ser748 was determined by immunoblot (IB) analysis using appropriate phospho-specific antibodies. Protein loading was confirmed using an anti-PKD antibody and 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 the potential roles of FHL1 and FHL2 in regulating the activation and functions of endogenous PKD in cardiac myocytes, we applied a loss-of-function approach and knocked down FHL1 or FHL2 protein expression by RNAi through transfection of NRVMs with synthetic siRNA duplexes specific for each FHL mRNA sequence. Transfection with FHL1 siRNA caused an approximately 75% decrease in FHL1 protein expression relative to control cells transfected with a scrambled siRNA sequence, with no apparent effect on FHL2 expression (Supplementary Figures S1A and S1B at http://www.biochemj.org/bj/457/bj4570451add.htm). Similarly, transfection with FHL2 siRNA caused a comparable decrease in FHL2 protein expression relative to cells transfected with a scrambled siRNA sequence, with no compensatory change in FHL1 expression (Supplementary Figures S1C and S1D). We then examined the consequences of the selective knockdown of each FHL isoform on PKD activation in response to stimulation with ET1 or PE. As illustrated in Figure 4, both stimuli significantly increased PKD autophosphorylation at Ser916 and transphosphorylation at Ser744/Ser748 in control cells transfected with scrambled siRNA. Knocking down FHL1 expression significantly attenuated the increase in PKD phosphorylation in response to ET1 (Figure 4A), but had no significant effect on the increase in PKD phosphorylation in response to PE (Figure 4B). In contrast, knockdown of FHL2 expression significantly attenuated the increases in PKD phosphorylation in response to both ET1 (Figure 4C) and PE (Figure 4D), with a greater inhibitory effect on the latter. These findings suggest that FHL1 and FHL2 facilitate PKD activation by multiple neurohormonal stimuli, potentially in a stimulus-dependent manner, by promoting transphosphorylation of the PKD activation loop. Interestingly, the effects of simultaneous knockdown of both FHL1 and FHL2 on PKD phosphorylation were similar to the effects of selective FHL2 knockdown, with no indication of an additive effect (Supplementary Figure S2 at http://www.biochemj.org/bj/457/bj4570451add.htm).


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 PKDNRVMs 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 μ M) (B and D) for 20 min. Phosphorylation status of endogenous PKD at Ser916 and Ser744/Ser748 was determined by immunoblot (IB) analysis using appropriate phospho-specific antibodies. Protein loading was confirmed using an anti-PKD antibody and 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

License
Show All Figures
getmorefigures.php?uid=PMC3927927&req=5

Figure 4: Effect of FHL1 or FHL2 knockdown on ET1- and PE-induced phosphorylation of endogenous PKDNRVMs 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 μ M) (B and D) for 20 min. Phosphorylation status of endogenous PKD at Ser916 and Ser744/Ser748 was determined by immunoblot (IB) analysis using appropriate phospho-specific antibodies. Protein loading was confirmed using an anti-PKD antibody and 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 the potential roles of FHL1 and FHL2 in regulating the activation and functions of endogenous PKD in cardiac myocytes, we applied a loss-of-function approach and knocked down FHL1 or FHL2 protein expression by RNAi through transfection of NRVMs with synthetic siRNA duplexes specific for each FHL mRNA sequence. Transfection with FHL1 siRNA caused an approximately 75% decrease in FHL1 protein expression relative to control cells transfected with a scrambled siRNA sequence, with no apparent effect on FHL2 expression (Supplementary Figures S1A and S1B at http://www.biochemj.org/bj/457/bj4570451add.htm). Similarly, transfection with FHL2 siRNA caused a comparable decrease in FHL2 protein expression relative to cells transfected with a scrambled siRNA sequence, with no compensatory change in FHL1 expression (Supplementary Figures S1C and S1D). We then examined the consequences of the selective knockdown of each FHL isoform on PKD activation in response to stimulation with ET1 or PE. As illustrated in Figure 4, both stimuli significantly increased PKD autophosphorylation at Ser916 and transphosphorylation at Ser744/Ser748 in control cells transfected with scrambled siRNA. Knocking down FHL1 expression significantly attenuated the increase in PKD phosphorylation in response to ET1 (Figure 4A), but had no significant effect on the increase in PKD phosphorylation in response to PE (Figure 4B). In contrast, knockdown of FHL2 expression significantly attenuated the increases in PKD phosphorylation in response to both ET1 (Figure 4C) and PE (Figure 4D), with a greater inhibitory effect on the latter. These findings suggest that FHL1 and FHL2 facilitate PKD activation by multiple neurohormonal stimuli, potentially in a stimulus-dependent manner, by promoting transphosphorylation of the PKD activation loop. Interestingly, the effects of simultaneous knockdown of both FHL1 and FHL2 on PKD phosphorylation were similar to the effects of selective FHL2 knockdown, with no indication of an additive effect (Supplementary Figure S2 at http://www.biochemj.org/bj/457/bj4570451add.htm).

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