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

Abstract

Protein kinase D (PKD) consists of a family of three structurally related enzymes that are co-expressed in the heart and have important roles in many biological responses. PKD1 is activated by pro-hypertrophic stimuli and has been implicated in adverse cardiac remodeling. Efforts to define the cardiac actions of PKD2 and PKD3 have been less successful at least in part because conventional methods provide a general screen for PKD activation but are poorly suited to resolve activation patterns for PKD2 or PKD3. This study uses Phos-tag SDS-PAGE, a method that exaggerates phosphorylation-dependent mobility shifts, to overcome this technical limitation. Phos-tag SDS-PAGE resolves PKD1 as distinct molecular species (indicative of pools of enzyme with distinct phosphorylation profiles) in unstimulated cardiac fibroblasts and cardiomyocytes; as a result, attempts to track PKD1 mobility shifts that result from agonist activation were only moderately successful. In contrast, PKD2 and PKD3 are recovered from resting cardiac fibroblasts and cardiomyocytes as single molecular species; both enzymes display robust mobility shifts in Phos-tag SDS-PAGE in response to treatment with sphingosine-1-phosphate, thrombin, PDGF, or H2O2. Studies with GF109203X implicate protein kinase C activity in the stimulus-dependent pathways that activate PKD2/PKD3 in both cardiac fibroblasts and cardiomyocytes. Studies with C3 toxin identify a novel role for Rho in the sphingosine-1-phosphate and thrombin receptor-dependent pathways that lead to the phosphorylation of PKD2/3 and the downstream substrate CREB in cardiomyocytes. In conclusion, Phos-tag SDS-PAGE provides a general screen for stimulus-specific changes in PKD2 and PKD3 phosphorylation and exposes a novel role for these enzymes in specific stress-dependent pathways that influence cardiac remodeling.

Protein kinase D3 regulates the expression of the immunosuppressive protein, PD‑L1, through STAT1/STAT3 signaling

“…ab128915; 1:20,000) from Abcam. Phos-tag SDS-PAGE (Wako) was performed according to a previously described protocol (23). Semi-quantitative analysis was performed by densitometry using Gel-Pro 32 software (version 3.1, Media Cybernetics, Bethesda).…”

Emergency Spatiotemporal Shift: The Response of Protein Kinase D to Stress Signals in the Cardiovascular System

“…The spatiotemporal dynamics of PKD activation also differ in adult cardiomyocytes: although phenylephrine (PE, an α1-AR agonist) and ET trigger comparable global PKD activation, PE induces transient sarcolemmal PKD recruitment and activation followed by nuclear import and ET prompts persistent sarcolemmal translocation and activity (Bossuyt et al, 2011). The PKD isoforms are also activated in an agonist-specific manner: norepinephrine (NE, an α/β-AR agonist) selectively activates PKD1 in neonatal myocytes and cardiac fibroblasts, whereas ET, thrombin and platelet derived growth factor (PDGF) favor PKD2/3 activation (Guo et al, 2011; Qiu and Steinberg, 2016). The molecular mechanisms underlying the differences between these two seemingly similar Gq-coupled receptors (GqR) remain to be identified.…”

“…Conversely, others examining global PKD1 activity reported suppression of PKD activity by β-AR agonists or PKA (Haworth et al, 2011; Sucharov et al, 2011). Some of these conflicting reports likely reflect that global PKD1 measurements do not necessarily capture discrete pools of PKD1 signaling (Qiu and Steinberg, 2016). Indeed, specific examination of PKD microdomain signaling revealed β-AR signaling triggers both local nuclear signaling and inhibits GqR-mediated PKD1 activation by preventing its intracellular translocation (Nichols et al, 2014).…”

Function and Regulation of Protein Kinase D in Oxidative Stress: A Tale of Isoforms

“…PKD3 was long thought not to be activated in oxidative stress because it lacks an N-terminal Tyr residue. However, PKD3 was recently shown to be activated via oxidative stress in fibroblasts, which is reversible by treatment with the PKC inhibitor GF109203X [ 82 ]. This indicates that PKC can phosphorylate both PKD2 and PKD3 without Tyr phosphorylation at the N-terminus during acute stress.…”