Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

The regulation of Na+/H+ exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. A fusion protein containing the regulatory domain of NHE1 was phosphorylated in vitro by wild-type PKD (PKD-WT), but not by the dominant-negative kinase-inactive mutant PKD-K618M. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells transfected with empty vector (control), PKD-WT or PKD-K618M together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux at pHi 7.0 (JH), was determined in single GFP-positive cells following intracellular acidification. Overexpression of PKD-WT had no effect on JH (3.78±0.24 mM/min v 3.48±0.25 mM/min in control). In contrast, overexpression of PKD-K618M increased JH (5.31±0.57 mM/min; p < 0.05 v control). Thus, native PKD appears to have a tonic inhibitory effect on NHE, which is relieved by the dominant-negative action of PKD-K618M. Exposure to phorbol ester significantly increased JH in control cells but failed to do so in cells overexpressing either PKD-WT (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal JH was already near-maximal). Overall, these results suggest that PKD inhibits NHE activity, and represents a novel pathway in the regulation of the exchanger.

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Presentation Number SAhaworth0246
Keywords: PKD, PKCµ, NHE1, COS-7, GFP