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

Jianfen Guo,Zoya Gertsberg,Nazira Ozgen,Abdelkarim Sabri,Susan F Steinberg

doi:10.1074/jbc.m110.208058

Jianfen Guo, Zoya Gertsberg + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.208058

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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 α(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 α(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.

Highlights

Institutes of Health Grants HL77860, HL-67101, and HL-28958. 1 To whom correspondence should be addressed: Dept. of Pharmacology, Protein kinase D (PKD)␮), PKD2 and PKD3) that play key roles in cell growth, differentiation, migration, and apoptosis (1)
PKD1 and PKD2 Are Activated in a Stimulus-specific Manner in Cardiomyocytes—We recently reported that the phosphorylated/activated form of PKD accumulates as a single molecular species, with an electrophoretic mobility corresponding to PKD1 (115 kDa), in cardiomyocytes treated with norepinephrine (NE), whereas the phosphorylated/activated form of PKD is resolved as doublet in cardiomyocytes treated with thrombin or PMA (12)
Activation loop phosphorylation was monitored with two phosphorylation-site specific antibodies (PSSAs) that recognize phosphorylation reactions in the conserved activation loop motifs of all three PKD isoforms; recent studies indicate that the anti-PKD-Ser(P)744/ Ser(P)748 PSSA primarily recognizes PKD1 phosphorylation at Ser744, whereas the anti-PKD1-Ser(P)748 PSSA preferentially recognizes PKD1 phosphorylation at Ser748 (14)

Summary

Introduction

Institutes of Health Grants HL77860, HL-67101, and HL-28958. 1 To whom correspondence should be addressed: Dept. of Pharmacology, PKD␮), PKD2 and PKD3 (or PKD␯)) that play key roles in cell growth, differentiation, migration, and apoptosis (1). We examined whether agonist-dependent pathways involving PKD1 or PKD2 increase CREB-Ser133 and/or cTnISer23/Ser24 phosphorylation in cardiomyocytes.

Results

Conclusion