The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

Yang Geng,Ahmet Hoke,Eric Delpire

doi:10.1074/jbc.m900142200

Abstract

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.

Highlights

Using a phosphopeptide-specific NKCC1 antibody, it was recently shown that NKCC1 phosphorylation instead of expression level increased in dorsal root ganglion (DRG) neurons upon nerve injury [1]
We chose to address the role of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1) by using an established nociceptive dorsal root ganglion sensory neuronal cell line [19], as well as isolated mouse DRG neurons
Using RT-PCR, four cation-chloride cotransporters (NCC1, KCC1, KCC3, and KCC4) as well as six protein kinases (SPAK, OSR1, WNK1, WNK2, WNK3, and WNK4) were identified in 50B11 cells, a pattern similar to that seen in native mouse and rat DRG neurons (Fig. 2)

Summary

Introduction

Our data demonstrate that both kinases are expressed to similar levels in both the cell line and isolated mouse DRG neurons, and that the kinases participate in concert to regulate NKCC1 function. Using RT-PCR, four cation-chloride cotransporters (NCC1, KCC1, KCC3, and KCC4) as well as six protein kinases (SPAK, OSR1, WNK1, WNK2, WNK3, and WNK4) were identified in 50B11 cells, a pattern similar to that seen in native mouse and rat DRG neurons (Fig. 2).

Results

Conclusion