Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 180–190 Aldosterone rapidly activates protein kinase D via a mineralocorticoid receptor/EGFR trans-activation pathway in the M1 kidney CCD cell line Victoria McEneaney, Brian J. Harvey, Warren Thomas ∗ Department of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Smurfit Building, Beaumont Hospital, Dublin 9, Ireland Received 20 December 2006; accepted 13 March 2007 Abstract Aldosterone elicits physiological responses through the modulation of gene expression and by stimulating signaling processes. Here we investigated the activation pathway of protein kinase D1 (PKD1) by aldosterone in the murine M1 renal cortical collecting duct cell line. Aldosterone stimulated a rapid increase in PKD1 activity peaking at 2–5 min and at 30 min after treatment that was insensitive to inhibitors of transcription or translation. PKD1 was not activated by aldosterone in MR null NIH-3T3 fibroblasts or M1-CCD cells propagated without dexamethasone, which did not express MR. PKD1 activation was sensitive to the MR antagonists spironolactone and RU28318 but not to the glucocorticoid receptor antagonist RU486. Aldosterone activation of PKD1 was inhibited by the epidermal growth factor (EGFR) antagonist tyrphostin AG1478 and by the c-Src inhibitor PP2. Western blotting revealed EGFR phosphorylation following aldosterone treatment at the c-Src tyrosine kinase-specific residue Tyr845. The activation of c-Src was dependent on its interaction with HSP84, since HSP84 antagonist 17-AAG inhibited both the phosphorylation of EGFR in response to aldosterone by c-Src and also the subsequent activation of PKD1. © 2007 Elsevier Ltd. All rights reserved. Keywords: Aldosterone; EGFR; PKC; Protein kinase D; Mineralocorticoid receptor 1. Introduction The mineralocorticoid aldosterone is a critical hormone in the regulation of sodium, potassium and proton fluxes across high resistance epithelia such as the renal cortical col- lecting duct (CCD) and the distal colon [1]. Aldosterone is released by the adrenal zona glomerulosa as the last stage in the renin-angiotensin system in response to decreased blood pressure or increased circulatory [K + ]. Aldosterone promotes Na + absorption and K + secretion in target tissues, addition- ally since water follows Na + by osmosis the net effect of aldosterone release is to increase extracellular fluid volume and consequently blood pressure [2]. Aldosterone not only modulates whole body, homeostatic, electrolyte balance but can also contribute to pathophysiological effects on renal tissue and the vasculature through the development of hyper- tension. Circulatory conditions associated with hypertension ∗ Corresponding author. Tel.: +353 1 809 3825; fax: +353 1 809 3778. E-mail address: wthomas@rcsi.ie (W. Thomas). are a major cause of death in the developed world. However clinical studies using antagonists of the intracellular miner- alocorticoid receptor (MR) have shown considerable benefit in severe congestive heart disease and after myocardial infarc- tion [3,4]. The classical mechanism of aldosterone action involves binding of aldosterone to the cytoplasmic MR that acts as a ligand-dependent transcription factor. MR interacts with the promoter sequences of aldosterone responsive genes to either promote or suppress their expression. The transcription lev- els of at least 63 genes expressed in the CCD [5] and 39 genes in the medullary collecting duct demonstrate changed expression levels following aldosterone treatment [6]. Rapid responses to aldosterone have been demonstrated in cellu- lar signaling pathways of different target tissues occurring within 15 min of hormone administration as reviewed in [7]. At least some of the rapid signaling events appear to be initiated by a receptor distinct from MR on the basis of exper- iments with cells derived from MR knockout mice [8]. In addition, cytosolic alkalinization of arterial cells treated with 0960-0760/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2007.03.043