The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase [MAPK] family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. These results provide an important role of CK2 in regulating nuclear ERK activities.Extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are central signaling proteins that mediate a variety of vital cellular processes, including proliferation, survival, and even apoptosis (1,4,15,29,50). In order to execute their functions, ERK molecules activate a large number of regulatory proteins, which are localized either in the cytoplasm or within various organelles, including mainly the nucleus (52). Indeed, the number of nuclear targets and downstream effectors of ERK, including a variety of transcription factors (17), is well over 100. These direct and indirect targets participate in the regulation of transcription as well as chromatin remodeling, and therefore they play a central role in mediating essentially all stimulated cellular processes (29). Moreover, because these ERK-induced nuclear activities are such central signaling processes, their dysregulation often leads to severe pathological processes, including oncogenic transformation, neurodegenerative diseases, and developmental diseases (15). In order to transmit their nuclear signals, ERK molecules that are localized in the cytoplasm of quiescent cells rapidly translocate into the nucleus upon stimulation. Although many details on ERK in the nucleus were already provided, the mechanisms of its translocation are not fully worked out yet.The nucleus is separated from the cytoplasm by a double membrane envelope (25). Nuclear shuttling of proteins occurs through a specialized nuclear pore complex (NPC), which ensures high selectivity of molecules for nuclear import/export, thus supporting proper cytoplasmic/nuclear molecular balance.

The RTK/Ras/Raf cascade is overactive in cancers. Its targets are the MAP kinases Erks, but Erks are not mutated in cancers. An active Erk, Erk1(R84S), is an oncoprotein. Further, Erk1(R84S) and Erk2(R65S) autophosphorylate the TEY motif and Thr-207/Thr-188. Erk2(R65S) autophosphorylates Thr-188 when dually mutated in the TEY.

The mechanisms regulating breast cancer differentiation state are poorly understood. Of particular interest are molecular regulators controlling the highly aggressive and poorly differentiated traits of basal-like breast carcinomas. Here we show that the Polycomb factor EZH2 maintains the differentiation state of basal-like breast cancer cells, and promotes the expression of progenitorassociated and basal-lineage genes. Specifically, EZH2 regulates the composition of basal-like breast cancer cell populations by promoting a "bi-lineage" differentiation state, in which cells co-express basal-and luminal-lineage markers. We show that human basal-like breast cancers contain a subpopulation of bi-lineage cells, and that EZH2-deficient cells give rise to tumors with a decreased proportion of such cells. Bi-lineage cells express genes that are active in normal luminal progenitors, and possess increased colony formation capacity, consistent with a primitive differentiation state. We found that GATA3, a driver of luminal differentiation, performs a function opposite to EZH2, acting to suppress bi-lineage identity and luminal-progenitor gene expression. GATA3 levels increase upon EZH2 silencing, mediating a decrease in bi-lineage cell numbers. Our findings reveal a novel role for EZH2 in controlling basal-like breast cancer differentiation state and intra-tumoral cell composition.

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