Abstract: | The regulation of plasma membraneNa+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel proteinkinase C- and phorbol ester-regulated kinase, was investigated. Todetermine the effect of PKD on NHE activity in vivo, intracellular pH(pHi) measurements were made inCOS-7 cells by microepifluorescence using the pH indicator cSNARF-1.Cells were transfected with empty vector (control), wild-type PKD, orits kinase-deficient mutant PKD-K618M, together with green fluorescentprotein (GFP). NHE activity, as reflected by the rate of acid efflux(JH), wasdetermined in single GFP-positive cells following intracellularacidification. Overexpression of wild-type PKD had no significanteffect on JH(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control atpHi 7.0). In contrast,overexpression of PKD-K618M increasedJH (5.31 ± 0.57 mM/min at pHi 7.0;P < 0.05 vs. control). Transfectionwith these constructs produced similar effects also in A-10 cells,indicating that native PKD may have an inhibitory effect on NHE in bothcell types, which is relieved by a dominant-negative action ofPKD-K618M. Exposure of COS-7 cells to phorbol ester significantlyincreased JH 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 JHwas already near maximal). A fusion protein containing the cytosolicregulatory domain (amino acids 637-815) of NHE1 (the ubiquitousNHE isoform) was phosphorylated in vitro by wild-type PKD, but with lowstoichiometry. These data suggest that PKD inhibits NHE activity,probably through an indirect mechanism, and represents a novel pathwayin the regulation of the exchanger. |