The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.. equally central to the regulation of cell signaling. The ubiquitin-proteasome pathway is an essential quality control mechanism directing degradation of mislocated, misfolded, and damaged proteins, and, by tempering the expression levels Carnosol of specific signaling proteins, it also exerts a level of control over cell physiology . Poly-ubiquitinated proteins, targeted by E3 ubiquitin ligases, can be acknowledged and degraded by the 26S proteasome, a multi-subunit, Carnosol multi-catalytic protease machine . Proteasome inhibitors have shown great promise as cancer therapeutics because they impact a variety of mechanisms affecting tumor cell proliferation and survival; proteasome inhibition interferes with cell cycle progression, upregulates tumor suppressors such as p53, and diminishes activation of pro-proliferation pathways such as those controlled by NFB and extracellular signal-regulated kinases (ERKs) , . The mitogen-activated protein kinases (MAPKs) ERK2/MAPK1 and ERK1/MAPK3 (hereafter referred to collectively as ERK1/2) are activated by phosphorylation in a canonical Raf MEK ERK kinase cascade in response to most growth factors and cytokines, and ERK1/2 phosphorylate more than 150 cytosolic and nuclear substrates , . Thus, they are grasp controllers of cell proliferation, differentiation, and migration. ERK signaling is usually inappropriately activated in a wide array of human cancers, which can be caused by an activating mutation in one of the upstream signaling proteins or through overexpression of growth factors or growth factor receptors , . The dual specificity phosphatases (DUSPs) have been linked to dephosphorylation of ERK1/2 and other MAPKs , and in many contexts, DUSP expression levels are known to be regulated through the ubiquitin-proteasome degradation pathway C. Accordingly, cells treated with MG132 or other proteasome inhibitors exhibit higher expression of MKP3/DUSP6, an ERK1/2-specific DUSP, accompanied by lower levels of ERK phosphorylation stimulated by growth factors C. Considering that a host of intracellular proteins are affected by proteasome inhibition, coupled with evidence that knockdown of MKP3 expression enhances growth factor-stimulated ERK phosphorylation in some contexts  but not in others , led us to question whether or not the diminution of ERK signaling in MG132-treated cells could be attributed solely to upregulation of MKP3 and other DUSPs. In this short paper, we confirm that MG132 treatment reduces phosphorylation of ERK in fibroblasts stimulated with platelet-derived growth factor (PDGF) or basic fibroblast growth factor (FGF) and show that this is usually caused by two parallel effects. For a given level of MEK activation, ERK phosphorylation is reduced, consistent with the proposed upregulation of ERK phosphatase activity, Carnosol but maximal MEK activation is also diminished. Materials and Methods Reagents Human recombinant PDGF-BB and murine recombinant FGF-2 were purchased from Peprotech (Rocky Hill, NJ). Antibodies against total ERK1/2, MEK1/2, Akt1/2/3 and MKP3 and phospho-specific antibodies against Rabbit Polyclonal to PHF1 PDGF -receptor pTyr751, Akt pSer473, ERK pThr202/pTyr204, and MEK pSer217/pSer221 were from Cell Signaling Technology (Beverly, MA). Antibodies against MKP1 were from Santa Cruz Biotechnology (Santa Cruz, CA). MG132 was purchased from Calbiochem (San Diego, CA) and aliquoted in DMSO; cells were incubated with the drug at a final concentration of 25 M, with an equivalent concentration of DMSO (0.2% v/v) serving as a vehicle control. All tissue culture reagents were from Invitrogen (Carlsbad, CA). Unless otherwise noted, all other reagents were from Sigma-Aldrich (St. Louis, MO). Cell Culture and Immunoblotting NIH 3T3 mouse fibroblast and HT-1080 human fibrosarcoma cell lines were acquired from American Type Culture Collection (Manassas, VA). Mouse embryonic fibroblasts, derived from pregnant CD-1 mice (Charles River Laboratories, Wilmington, MA), were.