In response to oncogenic activation, cells initially undergo proliferation followed by an irreversible growth arrest called oncogene-induced senescence (OIS), an endogenous defense mechanism against tumorigenesis. Oncogenic activation of ERK1/2 is essential for both the initial phase of cellular proliferation as well as subsequent premature senescence, but little is known about the specific contribution of ERK1 versus 2 to OIS. Here we show that depletion of ERK2 but not ERK1 by shRNA knockdown in MEFs leads to continuous proliferation bypassing senescence even in the presence of oncogenic HRAS(V12). Upon depletion of ERK2, induction of both p19(Arf) and p16(Ink4a) was significantly compromised after oncogenic HRAS(V12) expression, attenuating activation of the key tumor suppressors p53 and pRb. Here we demonstrate that ERK2 but not ERK1 indirectly regulates p19(Arf) and p16(Ink4a) both at the transcriptional and translational level. Oncogenic Ras expression after ERK2 knockdown downregulates Fra-1 and c-Jun, components of the activator protein-1 (AP-1) heterodimer essential for transactivation of p19(Arf). Similarly we show a significant decrease in the activation of p38 MAPK and ETS family members which are involved in the induction of p16(Ink4a). The role of ERK2 in translational regulation is observed by the lack of tuberin (TSC2) and p70 ribosomal S6 kinase 1 (p70S6K1) phosphorylation, components of the mTOR pathway, which enhances p19(Arf) mRNA translation during oncogenic Ras-induced senescence. These observations suggest that ERK2 but not ERK1 contributes to upregulation of p19(Arf) and p16(Ink4a) in a transcription- and translation-dependent manner during oncogenic Ras-induced senescence. Taken together, our data indicate that ERK2 is the key ERK isoform mediating the senescence signaling pathway downstream of oncogenic Ras.
Keywords: AP-1; Dmp1; EMT; ERK; ERK2; GFP; MEF; MEK; NS; OIS; Oncogenic Ras; PI3K; PML; SA-β-Gal; Senescence; TSC2; activator protein-1; cyclin d-interacting Myb-like protein 1; epithelial-to-mesenchymal transformation; extracellular signal-regulated kinase; green fluorescence protein; mTOR; mammalian target of rapamycin; mitogen-activated protein kinase kinase; mouse embryonic fibroblast; non-silencing; oncogene-induced senescence; p16(Ink4a); p19(Arf); p70 ribosomal S6 kinase 1; p70S6K1; pRb; phosphatidylinositol 3-kinase; phosphorylated histone H2AX; promyelocytic leukemia; retinoblastoma tumor suppressor protein; senescence associated β-galactosidase; shRNA; short hairpin RNA; tuberous sclerosis 2 or tuberin; γ-H2AX.