WNK1 Activates ERK5 by an MEKK2/3-dependent Mechanism

Bing-E Xu,Steve Stippec,Lisa Lenertz,Byung-Hoon Lee,Wei Zhang,Youn-Kyoung Lee,Melanie H Cobb

doi:10.1074/jbc.m313465200

Abstract

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.

Highlights

More than 500 protein kinases have been recognized in the human genome, ϳ1.7% of all human genes [1, 2]
To determine whether WNK1 acts upstream or downstream of MEKK2/3 in the ERK5 pathway, we examined the effects of dominant negative mutants of MEKK2/3 on ERK5 activation by WNK1
In the ERK5 pathway: 1) its activation of ERK5 can be blocked by kinase-dead mutants of MEKK2/3; 2) WNK1 interacts with MEKK2/3 in cells; 3) WNK1 phosphorylates MEKK2/3 in vitro; and 4) coexpression of WNK1 with MEKK3 activates it in cells

Summary

Introduction

More than 500 protein kinases have been recognized in the human genome, ϳ1.7% of all human genes [1, 2]. WNK4 has been shown to inhibit the activity of the sodium chloride cotransporter by reducing its membrane expression [11, 12]. This inhibition was reported to be dependent on the catalytic activity of WNK4, and the disease-causing mutation Q562E has less inhibitory effect. MEKK2 and MEKK3 are two closely related MAP3Ks with extremely high sequence identity within their catalytic domains, less so within their N-terminal regulatory domains [31] Both kinases have been shown to interact with MEK5 directly and activate the MEK5-ERK5 pathway [18, 19].

Methods

Results

Conclusion