KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

Abstract

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1^−/− colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

Introduction

Many cellular responses to external stimuli utilize mitogen-activated protein kinase (MAPK) pathways to carry out a diverse range of biological processes. Activation of these pathways, which are conserved in all eukaryotes, is often initiated by GTPases downstream of cell surface receptors, followed by sequential signal transduction through a three-component kinase system. One particular MAPK module consists of the protein kinases Raf, MEK, and ERK. Canonical activation of the Raf/MEK/ERK cascade occurs downstream of the small GTPase Ras to elicit a variety of cellular responses including proliferation, differentiation, and cell survival [1], [2], [3], [4]. Since the ERK pathway is integral for many cellular events, and constitutive pathway activation is frequently concurrent with many cancers, understanding the precise mechanisms that contribute to pathway activation are essential for developing therapeutic targets that modulate this pathway [5]. Kinase suppressor of Ras-1 (KSR1), first identified through genetic screens in Drosophila melanogaster and Caenorhabditis elegans, is an evolutionarily conserved protein that positively regulates the Raf/MEK/ERK cascade by functioning either upstream or in parallel with Raf-1 [6], [7], [8]. KSR1 functions as a molecular scaffold by binding several signaling components of the ERK cascade; and thus can enhance MAPK activation by regulating the efficiency of these interactions [9], [10], [11]. In addition to its scaffolding role, there is evidence that KSR1 functions as a protein kinase. The KSR1 C-terminus contains the eleven subdomains that are conserved in all protein kinases including the conserved aspartic acid and asparagine residues within subdomain VIb (HRDLKxxN motif) and the aspartic acid in subdomain VII (DFG motif) [12], [13]. However, the catalytic function of KSR1 remains controversial since mammalian KSR1 contains an arginine in place of the invariant lysine residue in subdomain II. This lysine positioned in subdomain II is involved in binding and orienting the ATP molecule to facilitate phosphotransfer of ATP γ-phosphate [14]. While lysine to arginine mutations in this position disrupt ATP binding and render many protein kinases inactive [15], [16], [17], [18], a KSR1 splice variant is able to bind ATP when the arginine was substituted with lysine or methionine [19]. This suggests that KSR1 might utilize a different lysine, as seen with the protein kinase with no lysine-1 (WNK1) [20], or may have a structurally unique ATP-binding cleft compared to other protein kinase domains. Therefore, further investigation into KSR1 catalytic function is warranted.

Initial reports of KSR1 protein kinase activity suggest that immunoprecipitated KSR1 autophosphorylates, as well as phosphorylates and activates Raf-1, in vitro [21], [22], [23]. However, immunoprecipitated KSR1 contains additional co-precipitating protein kinases making it difficult to delineate KSR1 protein kinase activity from that of other contaminating kinases in the assay [24], [25]. Therefore, to resolve KSR1 kinase activity from other protein kinases in vitro requires isolating recombinant proteins expressed in a system with no known serine/threonine protein kinases, such as Eschrichia coli [26].

Here we report that bacterially-derived KSR1 underwent serine autophosphorylation, phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MEK1 (rMEK K97M). We also demonstrate that both a functional KSR1 kinase domain and MEK protein kinase activity are required for resistance to TNF-induced cell death in colon epithelial cells. Taken together, these data indicate that in addition to a scaffold, KSR1 is indeed a functional protein kinase in the ERK pathway downstream of TNF signaling.