Protein kinase D inhibits plasma membrane Na(+)/H(+) exchanger activity.
The regulation of plasma membrane Na(+)/H(+) exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pH(i)) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux (J(H)), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J(H) (3. 48 +/- 0.25 vs. 3.78 +/- 0.24 mM/min in control at pH(i) 7.0). In contrast, overexpression of PKD-K618M increased J(H) (5.31 +/- 0.57 mM/min at pH(i) 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J(H) in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J(H) was already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637-815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.
Haworth
R S
RS
Centre for Cardiovascular Biology and Medicine, King's College London, London, United Kingdom.
Sinnett-Smith
J
J
Rozengurt
E
E
Avkiran
M
M
D000818:Animals; D001011:Aorta / Q000166:cytology; D001578:Benzopyrans; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D005456:Fluorescent Dyes; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000201:enzymology; D016296:Mutagenesis / Q000502:physiology; D009284:Naphthols; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D001578:Benzopyrans; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; C068301:carboxy-seminaphthorhodaminefluoride; D049452:Green Fluorescent Proteins; C089212:protein kinase D; D011493:Protein Kinase C