Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Sep 23;286(38):33390-400.
doi: 10.1074/jbc.M111.246447. Epub 2011 Jul 27.

Spatiotemporally distinct protein kinase D activation in adult cardiomyocytes in response to phenylephrine and endothelin

Julie Bossuyt  1 , Chia-Wei Chang, Kathryn Helmstadter, Maya T Kunkel, Alexandra C Newton, Kenneth S Campbell, Jody L Martin, Sven Bossuyt, Seth L Robia, Donald M Bers
Affiliations

Spatiotemporally distinct protein kinase D activation in adult cardiomyocytes in response to phenylephrine and endothelin

Julie Bossuyt et al. J Biol Chem. .

Abstract

Protein kinase D (PKD) is a nodal point in cardiac hypertrophic signaling. It triggers nuclear export of class II histone deacetylase (HDAC) and regulates transcription. Although this pathway is thought to be critical in cardiac hypertrophy and heart failure, little is known about spatiotemporal aspects of PKD activation at the myocyte level. Here, we demonstrate that in adult cardiomyocytes two important neurohumoral stimuli that induce hypertrophy, endothelin-1 (ET1) and phenylephrine (PE), trigger comparable global PKD activation and HDAC5 nuclear export, but via divergent spatiotemporal PKD signals. PE-induced HDAC5 export is entirely PKD-dependent, involving fleeting sarcolemmal PKD translocation (for activation) and very rapid subsequent nuclear import. In contrast, ET1 recruits and activates PKD that remains predominantly sarcolemmal. This explains why PE-induced nuclear HDAC5 export in myocytes is totally PKD-dependent, whereas ET1-induced HDAC5 export depends more prominently on InsP(3) and CaMKII signaling. Thus α-adrenergic and ET-1 receptor signaling via PKD in adult myocytes feature dramatic differences in cellular localization and translocation in mediating hypertrophic signaling. This raises new opportunities for targeted therapeutic intervention into distinct limbs of this hypertrophic signaling pathway.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Agonist-dependent signaling to HDAC5. a, signaling pathway to HDAC5 nuclear export. G protein-coupled receptor (GPCR) activation leads to InsP3 production, which in turn causes local, nuclear Ca2+ release and CaMKII/PKD activation. These kinases then phosphorylate HDAC5 triggering its nuclear export. b, rabbit ventricular myocyte expressing GFP-HDAC5 exposed to 10 μm PE, with enlargements of the nucleus 2 (N2) region before and after a 60-min PE exposure. HDAC5 nuclear export was analyzed as a decrease of Fnuc/Fcyto normalized to the initial ratio (n = 12 for 100 nm ET1, 20 for 10 μm PE). c, myocytes were pretreated with 10 μm BisI or Gö6976 for 20–30 min before ET1 or PE exposure for 60 min. d, myocytes were pretreated with 1 μm KN93 or 2 μm 2-APB for 20–30 min prior to ET1 or PE exposure (***, p < 0.001).
FIGURE 2.
FIGURE 2.
Assessment of global PKD activation. a, representative Western blots of PKD expression and phosphorylation (using global and phosphospecific antibodies) in adult rabbit myocytes and quantified protein signals (n = 6). b, principle of DKAR measurements of PKD activity (adapted from Kunkel et al. (25)), upon phosphorylation of the substrate sequence a molecular switch occurs resulting in a decrease of FRET. Expression of the sensor is shown in rabbit myocytes with selective excitation of CFP, YFP, or FRET (excitation of CFP, detection of YFP emission). c, ratiometric real time measurements of DKAR FRET in adult rabbit myocytes (also expressing PKD1, n = 6). d, acceptor photobleach-induced enhancement of CFP fluorescence (DKAR FRET) after 20 min exposure to PE, ET1, or PDBu (n = 6). Data are presented as the % change obtained, assuming that fully phosphorylated DKAR depicts no basal FRET.
FIGURE 3.
FIGURE 3.
Agonist-dependent spatiotemporal dynamics of PKD1 localization. a, rabbit ventricular myocyte expressing PKD1-GFP exposed to PDBu (top left), ET1 (middle left), and PE (bottom left). Right panels show immunolocalization of endogenous PKD1 in non-transfected myocytes. Right bottom panels show (b) PKD1-GFP localization was analyzed as FSL/Fcyto and Fnuc/Fcyto for membrane recruitment and nuclear import, respectively (n ≥ 5; all 3 curves were significantly different (left) and ET1 was different from PE and PDBu (right) by analysis of variance). c, for analysis of recruitment to T-tubular membrane (or Z-line), plot profiles were fit to a sine wave (left) and amplitude was taken as Z-line signal (right, ET1 was different from PE by analysis of variance)).
FIGURE 4.
FIGURE 4.
TIRF measurements of PKD1-GFP. a, rabbit ventricular myocyte expressing PKD1-GFP is shown using epifluorescence (top) and TIRF imaging (lower panels), including membrane recruitment in TIRF mode upon exposure to PDBu. b, agonist-dependent PKD1 membrane recruitment analyzed as increases in GFP fluorescence (normalized to initial signal) in TIRF mode (n = 10; all 4 curves were significantly different by analysis of variance), including the expanded display of first 75 s (c) and amplitude-normalized time course (d).
FIGURE 5.
FIGURE 5.
FRAP analysis of PKD membrane association. a, fluorescence recovery after spot photobleaching in TIRF mode. Target site was bleached after steady-state was reached for agonist-dependent membrane recruitment (n = 8). b, FRAP portion of signals in a, normalized to steady state recovery (t½ for PE significantly different versus Ctl and also versus ET and PDBu). c, selective ablation of fluorescence at the target site (red arrow or dark spot in ratio image) is shown in TIRF images at the left and middle. Right panel is a three-dimensional isosurface rendering of the bleach spot. d, spatial profile of bleach spot, which is plotted as fluorescence versus distance across target site (squares, data points; lines, Gaussian fit/simulation). Recovery due to membrane (lateral) diffusion is seen as change in width, whereas recovery from the cytosol (PKD turnover) is seen as a change in volume. e, extraction of the width component of FRAP recovery (indicative of lateral membrane diffusion) and f, of the volume component (indicative of exchange with cytosol).
FIGURE 6.
FIGURE 6.
Agonist-dependent activation of nuclear and sarcolemmal PKD activity. a, PKD activity measured with sarcolemmal-targeted DKAR-SL. Left, acceptor photobleach-induced enhancement of donor fluorescence (FRET after 20 min agonist treatment, n = 8). Right, rabbit myocyte DKAR-SL localization. b, PKD activity measured with nuclear-targeted DKAR-Nuc. Left, acceptor photobleach measurement of FRET after 60 min agonist. Right, rabbit myocyte DKAR-Nuc localization. c, ratiometric, real time measurement of nuclear or membrane PKD activity (arrow indicates drug addition) (n = 6, PE curve and ET1 curve differ significantly for DKAR-Nuc, but not for DKAR-SL). *, p < 0.05 versus control.
FIGURE 7.
FIGURE 7.
Working hypothesis of PKD activation. PE shifts PKD to the sarcolemma where it is activated and rapidly translocates to the nucleus (rapid membrane-cytosolic movement). ET1 (like PDBu) causes sarcolemmal PKD recruitment and activation, but PKD remains largely membrane bound, with only slow and small nuclear translocation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Frey N., Olson E. N. (2003) Annu. Rev. Physiol. 65, 45–79 - PubMed
    1. McKinsey T. A. (2007) Cardiovasc. Res. 73, 667–677 - PubMed
    1. Avkiran M., Rowland A. J., Cuello F., Haworth R. S. (2008) Circ. Res. 102, 157–163 - PubMed
    1. Backs J., Backs T., Bezprozvannaya S., McKinsey T. A., Olson E. N. (2008) Mol. Cell. Biol. 28, 3437–3445 - PMC - PubMed
    1. Fielitz J., Kim M. S., Shelton J. M., Qi X., Hill J. A., Richardson J. A., Bassel-Duby R., Olson E. N. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 3059–3063 - PMC - PubMed

Publication types

MeSH terms