title: 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.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Goettel
authors.forename: Dongchun
authors.initials: D
authors.lastname: Liang
authors.forename: Valda C
authors.initials: VC
authors.lastname: Hilliard
authors.forename: Karen L
authors.initials: KL
authors.lastname: Edelblum
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Broadus
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Gould
authors.forename: Steven K
authors.initials: SK
authors.lastname: Hanks
authors.forename: D Brent
authors.initials: DB
authors.lastname: Polk
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002384:Catalysis; D003106:Colon / Q000166:cytology; D004847:Epithelial Cells; D029968:Escherichia coli Proteins; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D029968:Escherichia coli Proteins; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D048369:MAP Kinase Kinase 1