KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1 |
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Authors: | Goettel Jeremy A Liang Dongchun Hilliard Valda C Edelblum Karen L Broadus Matthew R Gould Kathleen L Hanks Steven K Polk D Brent |
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Affiliation: | aDepartment of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA;bDepartment of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232, USA;cHoward Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232, USA |
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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. |
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Keywords: | Abbreviations: MAPK, mitogen-activated protein kinase ERK, extracellular signal-regulated kinase KSR1, kinase suppressor of Ras-1 MEK, MAPK/ERK kinase MBP, myelin basic protein TNF, tumor necrosis factor WNK1, with no lysine-1 GFP, green fluorescent protein EGF, epidermal growth factor YAMC, young adult mouse colon TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling PP2A, protein phosphatase 2A MEKK1, MEK kinase-1 |
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