Protein Kinase D Isoforms Are Activated in an Agonist-specific Manner in Cardiomyocytes

Abstract

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The ␣ 1 -adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the ␣ 1 -adrenergic receptor pathway that activates PKD1 and the thrombin-and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser 133 and cTnI-Ser 23 /Ser 24 phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser 133 phosphorylation; there is no associated increase in cTnI-Ser 23 /Ser 24 phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser 133 phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure. Protein kinase D (PKD)2 consists of a family of three structurally related serine-threonine kinases (termed PKD1 (or PKD), PKD2 and PKD3 (or PKD)) that play key roles in cell growth, differentiation, migration, and apoptosis (1). PKD isoforms share a common domain structure, consisting of a C-terminal kinase domain and an N-terminal regulatory domain. The regulatory domain contains a C1 domain that anchors full-length PKD to diacylglycerol/phorbol ester-containing membranes and a pleckstrin homology (PH) domain that participates in intramolecular autoinhibitory interactions. PKD activation is generally attributed to a phosphorylation-dependent mechanism involving PKC. Receptors that activate phospholipase C and promote diacyglycerol accumulation co-localize PKD with allosterically activated PKC at lipid membranes. PKD is then activated as a result of phosphorylation at Ser 744 and Ser 748 , a pair of highly conserved serine residues in the activation loop of the kinase domain (nomenclature based upon rodent PKD1). Once activated, PKD1 and PKD2 execute autophosphorylation reactions at a serine residue in a PKD consensus phosphorylation motif at the extreme C terminus (Ser 916 in...

β-Adrenergic Signaling Inhibits G q -Dependent Protein Kinase D Activation by Preventing Protein Kinase D Translocation

“…The activation is largely dependent on phosphorylation of the activation loop residues (S744/748 in murine numbering) by both PKC-dependent and -independent mechanisms. 23–26 Despite this general consensus on global PKD activation for GPCR agonists, our group 27, 28 recently identified dramatic spatiotemporal differences in PKD1 activation for two G q -coupled agonists, phenylephrine (PE) and endothelin-1 (ET1) in adult cardiomyocytes. While both agonists triggered comparable global PKD1 activation, PE induced fleeting sarcolemmal PKD1 translocation and activation followed by nuclear translocation and ET1 prompted persistent sarcolemmal recruitment and activity.…”

Regulation of Protein Kinase D1 Activity

“…Rather, studies in cardiomyocytes identify stimulus-specific differences in PKD1 activation by ␣ 1 -adrenergic receptors (␣ 1 -ARs) and endothelin-1 receptors, two seemingly similar G q -coupled receptors. Here, ␣ 1 -ARs induce a rapid increase in PKD1 activity that is sustained for at least 1 h; the rapid and sustained phases of ␣ 1 -AR-dependent PKD1 activation both require PKC activity (Guo et al, 2011). In contrast, endothelin-1 receptors induce a transient PKC-dependent increase in PKD1 activity that is followed by a more sustained increase in PKD1 that does not require PKC activity (Guo et al, 2011).…”

“…Alternatively, a secondary Ser 742 autophosphorylation defect induced by the S910A substitution regulates these processes; phosphorylation at the homologous activation loop site in D kinase family-2 (a PKDrelated enzyme in Caenorhabditis elegans) enhances enzyme stability (Feng et al, 2007 is a target for autocatalytic phosphorylation, these results suggest that PKD1-Ser 742 phosphorylation in vivo is mediated primarily by a cis-autophosphorylation reaction that is defective in the catalytically inactive enzyme (Rybin et al, 2009). Second, there is evidence that certain G protein-coupled receptor agonists induce a rapid/coordinate PKC-dependent increase in PKD1 phosphorylation at Ser 738 /Ser 742 that is followed by a more sustained increase in PKD1 phosphorylation at Ser 742 ; Ser 742 phosphorylation during the late phase of G protein-coupled receptor activation occurs via an autocatalytic mechanism that does not require PKC activity (Jacamo et al, 2008;Guo et al, 2011). This sustained GPCRdependent mechanism for PKD1 activation loop phosphorylation (via an autocatalytic mechanism that does not require PKC activity) promotes extracellular signal-regulated kinase activation and mitogenic signaling in some cell types (Sinnett- Smith et al, 2009).…”

“…Here, ␣ 1 -ARs induce a rapid increase in PKD1 activity that is sustained for at least 1 h; the rapid and sustained phases of ␣ 1 -AR-dependent PKD1 activation both require PKC activity (Guo et al, 2011). In contrast, endothelin-1 receptors induce a transient PKC-dependent increase in PKD1 activity that is followed by a more sustained increase in PKD1 that does not require PKC activity (Guo et al, 2011). This PKC-independent mechanism for PKD1 activation may have evolved to support signaling responses at late time points, when PKC isoforms are down-regulated.…”

Phos-tag SDS-PAGE resolves agonist- and isoform-specific activation patterns for PKD2 and PKD3 in cardiomyocytes and cardiac fibroblasts

“…nPKCs then trans -phosphorylate PKD at conserved serine residues in the activation loop. However, PKC-independent activation loop autophosphorylation also can contribute to PKD1 activation under specific stimulatory conditions [4, 5]. Activated forms of PKD1 and PKD2 (but not PKD3) then autophosphorylate at a PKD consensus phosphorylation motif (in a PDZ domain-binding motif) at the extreme C-terminus - this modification regulates interactions with scaffolding proteins, trafficking to distinct cellular subdomains, and the amplitude/tempo of PKD signaling responses [6, 7].…”