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 E. 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.

Signal control through Raf: in sickness and in health

“…A functional catalytic lysine appears to be absent from KSR, unlike what we found in WNK protein kinases; in WNKs the catalytic lysine is not in the canonical position, but it is nearby in a distinct structural element [33]. Nevertheless, KSR1 purified from E. coli can directly phosphorylate MEK1 in vitro, albeit at a rate that may be representative of a catalytically impaired Raf mutant [29]. Regardless, KSR is a critical component of the MAPK pathway and thus subject to modulation just as are the other proteins in the pathway.…”

“…Glucose-induced insulin gene transcription has been shown to require a set of core transcription factors, including BETA2 (also known as NeuroD1), PDX1, MafA, and NFAT [29,[40][41][42][43][44][45][46]. We showed that these transcription factors act synergistically to promote insulin gene transcription in an ERK1/2-dependent manner, as phosphorylation of both Beta2 and PDX-1 is direct [47].…”

“…The relevance of the catalytic activity of KSR proteins is in debate [27][28][29][30][31]. KSRs contain arginine instead of the invariant catalytic lysine in the N-terminal region of the kinase domain present in other protein kinases.…”

Comparative Oncogenomics Implicates the Neurofibromin 1 Gene (NF1) as a Breast Cancer Driver

“…Omg is expressed primarily in neural tissues and is required for myelination in the central nervous system. Ksr1 promotes oncogenic RAS and MAPK signaling in mice and cells, so its deletion would be expected to actually inhibit tumor growth (Lozano et al 2003;Goettel et al 2011). WSB1 appears to participate in an E3 ubiquitin ligase complex not known to be associated with cancers.…”

Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF

“…At that time, when the KSR1 function was considered only in scaffolding of MAPK components, the loss of signalling at supra-physiologic KSR1 expression levels was proposed to be due to the titration of pathway members away from each other, a phenomenon termed as "combinatorial inhibition" [54]. However, emerging evidence suggests that KSR1 possesses dual activity as a scaffold protein and maybe also as active kinase phosphorylating C-RAF and MEK [17,[57][58][59]. Moreover, KSR1 can also form dimers with RAF and modulate its activity adding complexity to the simple view of KSR1 scaffolding function [17][18].…”