Protein Kinase D Links Gq-coupled Receptors to cAMP Response Element-binding Protein (CREB)-Ser133 Phosphorylation in the Heart

Abstract

Extracellular ligands stimulate cardiac growth and differentiation by activating a network of protein kinases that phosphorylate transcription factors and alter gene expression. Many of these mechanisms are resurrected in the damaged or failing heart in an attempt to compensate for contractile dysfunction. Our previous studies focused on the cellular actions of thrombin, a serine protease that is generated at sites of cardiac injury and proteolytically activates protease-activated receptor-1 (PAR-1), 3 a G protein-coupled receptor that activates a spectrum of effectors that contribute to cardiac fibroblast proliferation and cardiomyocyte hypertrophy (1). Certain aspects of PAR-1 signaling are cell-specific; PAR-1 activates ERK primarily via an epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts and a distinct pathway that does not require EGFR kinase activity in cardiomyocytes. Of note, the PAR-1 signaling pathway in cardiomyocytes triggers a form of cellular remodeling that resembles the changes observed in dilated cardiomyopathies (with pronounced cell elongation and relatively little increased cell width). This hypertrophic phenotype is morphologically distinct from the concentric hypertrophy induced by ␣ 1 -AR agonists such as norepinephrine (NE) or P. multocida toxin (PMT, a direct G␣ q agonist); NE and PMT induce very pronounced increases in overall cell size in association with enhanced sarcomeric organization and atrial natriuretic factor expression (2). cAMP response element-binding protein (CREB) is a bZip transcription factor that forms homo-or heterodimers with itself or with other CREB/ATF family members and binds to specific DNA elements (termed cAMP response elements or CREs) within the regulatory regions of CREB target genes. CREB has been implicated in the maintenance of normal ventricular structure and function; cardiac-specific overexpression of dominant-negative CREB leads to dilated cardiomyopathy and interstitial fibrosis (3). CREB also has been implicated in the electrophysiological remodeling that accompanies pacinginduced cardiac memory in dogs (4). CREB is regulated via phosphorylation at Ser 133 , which activates CREB-dependent gene transcription by recruiting a coactivator (CREB-binding protein, or CBP) to the promoters of CREB target genes.* This work was supported, in whole or in part, by National Institutes of Health Grants HL77860, HL-67101, and HL-28958. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Reactive Oxygen Species Decrease cAMP Response Element Binding Protein Expression in Cardiomyocytes via a Protein Kinase D1-Dependent Mechanism That Does Not Require Ser133 Phosphorylation

“…Cardiomyocytes were isolated from the hearts of 2-day-old Wistar rats by a trypsin dispersion procedure using a differential attachment procedure to enrich for cardiomyocytes followed by irradiation as detailed in previous publications (Steinberg et al, 1991;Ozgen et al, 2008). The final yield of cells was typically 2.5 to 3 ϫ 10 6 per neonatal heart.…”

“…pCRE-luciferase (Stratagene, La Jolla, CA) and Renilla reniformis luciferase (Promega, Madison, WI) vectors were introduced into cardiomyocytes using a nucleofector kit (Amaxa Biosystems, Gaithersburg, MD) according to manufacturers' instructions. Cre-luciferase signals were normalized to luminescence from R. reniformis (to eliminate interassay variability and control for agonist-dependent changes in transfection efficiency or cell viability) using a dual-luciferase reporter assay system (Promega) according to methods described previously (Ozgen et al, 2008 also activates PKD, a family of three structurally related serine/threonine kinases (PKD1, PKD2, and PKD3) that recently also have been implicated as CREB-Ser 133 kinases (Johannessen et al, 2007;Ozgen et al, 2008). PKD activation is generally attributed to a phosphorylation-dependent mechanism involving protein kinase C (PKC).…”

“…Western blotting was performed on cell extracts according to methods described previously or the manufacturer's instructions (Ozgen et al, 2008;Rybin et al, 2009 (Danvers, MA). Studies that validate the specificity of the phospho-site-specific antibodies (PSSAs) that recognize the phosphorylated forms of PKD1 have been published previously (Jacamo et al, 2008;Rybin et al, 2009).…”

Protein Kinase D1 Autophosphorylation via Distinct Mechanisms at Ser744/Ser748 and Ser916

“…Terry Rogers and William Randall, University of Maryland) or ␤-galactosidase as a control. Infections were performed on cultures grown for 5 days in minimum Eagle's medium supplemented with 10% fetal calf serum (with protein extracts prepared 48 h following infections) according to methods described previously (6).…”

“…However, the list of known PKD substrates remains relatively short. We and others recently implicated PKD as a CREB-Ser 133 kinase that regulates Cre-dependent transcriptional responses (6,7). PKD also functions as a physiologically relevant HDAC5 kinase (8).…”