A cellular threshold for active ERK1/2 levels determines Raf/MEK/ERK-mediated growth arrest versus death responses

Abstract

In addition to its conventional role for cell proliferation and survival, the Raf/MEK/Extracellular signal-regulated kinase (ERK) pathway can also induce growth arrest and death responses, if aberrantly activated. Here, we determined a molecular basis of ERK1/2 signaling that underlies these growth inhibitory physiological outputs. We found that overexpression of ERK1 or ERK2 switches ΔRaf-1:ER-induced growth arrest responses to caspase-dependent apoptotic death responses in different cell types. These death responses, however, were reverted to growth arrest responses upon titration of cellular phospho-ERK1/2 levels by the MEK1/2 inhibitor AZD6244. These data suggest that a cellular threshold for active ERK1/2 levels exists and affects the cell fate between death and growth arrest. We also found that death-mediating ability of ERK2 is abolished by the catalytic site-disabling Lys52Arg replacement or significantly attenuated by the F-site recruitment site-disabling Tyr261Asn replacement, although unaffected by the mutations that disable the common docking groove or the dimerization interface. Therefore, ERK1/2 mediates death signaling dependently of kinase activity and specific physical interactions. Intriguingly, Tyr261Asn-replaced ERK2 could still mediate growth arrest signaling, further contrasting the molecular basis of ERK1/2-mediated growth arrest and death signaling. These data reveal a mechanism underlying the role of ERK1/2 as a focal point of Raf/MEK/ERK-mediated growth arrest and death signaling.

Growth Inhibitory Signaling of the Raf/MEK/ERK Pathway

“…Consistent with our observation, other groups showed that overexpression of ectopic ERK1/2 can induce robust cell death responses in a subset of human B-Raf V600E melanoma cells [59,60]. Kinase function of ERK1/2 is crucial for these death effects, as catalytic site-disabled ERK2 mutants cannot induce cell death responses [58]. These phenomena suggest that the magnitude of ERK1/2 catalytic activity should be higher than a certain threshold to trigger cell death, while the availability of their death-specific substrates is also important in determining the cell fate.…”

“…Intriguingly, mutations that impair the CD site did not affect the growth arrest responses induced by ERK2-L73P/S151D or the aforementioned death responses induced by wild type ERK2 overexpression (Section 2.3) in Raf/MEK-activated cells [58,74]. In contrast, the FRS (Y261N) mutation markedly attenuated the death responses induced by wild-type ERK2 overexpression in Raf/MEK-activated cells [58], suggesting the significance of FRS in ERK1/2-mediated growth inhibitory signaling; the effects of Y261N could not be evaluated in ERK2-L73P/S151D because Y261N inhibited autophosphorylation of this mutant [74]. The F-site signature "Phe-Xaa-Phe-Pro" is relatively less frequent than the D-site signature and is found only in certain ERK1/2 substrates, including the cell-proliferative transcription factors ELK1, c-Fos, Fra1, and c-Myc, as well as the anti-apoptotic BH3-only protein BimEL [76][77][78].…”

“…ERK1 and ERK2 are also functionally redundant and interchangeable in Raf/MEK/ERK-mediated growth inhibitory signaling [43]. Intriguingly, we recently demonstrated that overexpression of ERK1 or ERK2 can switch C-Raf-induced growth arrest responses to caspase-dependent apoptotic death responses in different cell line models, which can then be reverted to growth arrest responses upon titrating the degree of ERK1/2 activation using MEK1/2 inhibitors [58]. Consistent with our observation, other groups showed that overexpression of ectopic ERK1/2 can induce robust cell death responses in a subset of human B-Raf V600E melanoma cells [59,60].…”

“…The common docking (CD) site and F-recruitment site (FRS) are two major domains of ERK1/2 for physical interactions [10], which are independent of each other with respect to ERK1/2 catalysis [75]. Intriguingly, mutations that impair the CD site did not affect the growth arrest responses induced by ERK2-L73P/S151D or the aforementioned death responses induced by wild type ERK2 overexpression (Section 2.3) in Raf/MEK-activated cells [58,74]. In contrast, the FRS (Y261N) mutation markedly attenuated the death responses induced by wild-type ERK2 overexpression in Raf/MEK-activated cells [58], suggesting the significance of FRS in ERK1/2-mediated growth inhibitory signaling; the effects of Y261N could not be evaluated in ERK2-L73P/S151D because Y261N inhibited autophosphorylation of this mutant [74].…”

Mortalin/HSPA9 targeting selectively induces KRAS tumor cell death by perturbing mitochondrial membrane permeability

“…Cell viability was determined by trypan blue exclusion assay (Invitrogen) or by flow cytometry of cells stained with the fluorescent DNA intercalator TO-PRO 3 (Invitrogen). The colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay was performed as previously described 19 . For cell cycle and death analyses, flow cytometry of cells stained with annexin V and propidium iodide (Invitrogen) was performed using the Guava EasyCyte flowcytometry system (MilliporeSigma, Billerica, MA, USA), as previously described 47 .…”