Research ArticleKSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
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.
Section snippets
Generation of stable KSR1 cell lines
The conditionally immortalized KSR1−/− colon epithelial cell line was generated by crossing a KSR1−/− mouse with the H-2Kb–tsA58 ImmortoMouse (Charles River Laboratories International Inc., Wilmington, MA), as previously described [23], [27], [28]. N-terminally FLAG-tagged murine wild-type KSR1 or murine kinase-inactive KSR1 harboring an amino acid substitution of aspartic acid to alanine at two residues within the kinase domain that are critical for enzymatic activity (D683A/D700A) were a
KSR1 promotes TNF-mediated cell survival through phosphorylation of MEK1
The pleiotropic pro-inflammatory cytokine, tumor necrosis factor, (TNF) elicits various cellular responses in a variety of cell types in vitro and in vivo and plays a pivotal role in chronic gastrointestinal disorders including celiac and inflammatory bowel diseases [30], [31], [32]. Previous studies from our lab show that the colon epithelium of KSR1−/− mice is sensitive to TNF-induced apoptosis compared to wild-type mice [23]. Therefore, to determine if KSR1 kinase activity is required to
Discussion
Our findings indicate that KSR1 is a functional protein kinase that, along with MEK1 protein kinase activity, is required for protection against TNF-induced cell death. The observation that MEK associated with wild-type KSR1 had increased phosphorylation, together with our in vitro kinase assays using recombinant proteins, suggests that KSR1 is capable of direct phosphorylation of MEK. Interestingly, while phosphorylation of rMEK in the MEK activation loop was detected for both recombinant and
Acknowledgments
We would like to thank Philip Dubé, Stuart Hobbs, and Fang Yan for their thoughtful discussions and critique of the manuscript. This work was supported by NIH grants GM068786 (K.L.G), DK066176 and DK56008 (D.B.P).
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