title: Active ERK2 is sufficient to mediate growth arrest and differentiation signaling.
abstract: Although extracellular signal-regulated kinases (ERK1/2) have been shown to be required in Raf/MEK/ERK pathway signaling, its sufficiency for mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously described ERK2 mutants that exhibit enhanced autophosphorylation of TEY sites in the activation loop in terms of their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. We demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp substitutions that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation, whereas expression of ERK2-I84A and ERK2-R65S/D319N is not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D only mildly increased ERK kinase activity in cells, as assessed using the ERK substrates p90(RSK) and ETS domain-containing protein (ELK1). However, ERK2-L73P/S151D expression effectively induced down-regulation of androgen receptors, Retinoblastoma (Rb) protein and E2F1 transcription factor, and up-regulation of p16(INK4A) and p21(CIP1), accompanied by cell-cycle arrest and morphological differentiation in LNCaP cells and neurite-like processes in PC12 cells. These effects and the TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introduction of the active site-disabling Lys52Arg mutation, suggesting that its autoactivation drives this signaling. Moreover, introduction of mutations Asp316/319Ala or Asp319Asn, which impair the common docking site/D-domain-based physical interaction of ERK, did not significantly affect ERK2-L73P/S151D signaling, suggesting that ERK2 mediates growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Thus, our study presents convincing evidence of ERK sufficiency for Raf/MEK/ERK signaling.
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.
authors: Pui-Kei
authors: PK
authors: Wu
authors: Seung-Keun
authors: SK
authors: Hong
authors: Seung-Hee
authors: SH
authors: Yoon
authors: Jong-In
authors: JI
authors: Park
mesh_terms: D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016922:Cellular Senescence; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D008297:Male; D009154:Mutation; D016716:PC12 Cells; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011944:Receptors, Androgen / Q000378:metabolism; D020218:Response Elements; D048490:raf Kinases / Q000378:metabolism
chemical_list: D011944:Receptors, Androgen; D048490:raf Kinases
keywords: ERK/MAPK; MEK; Raf; differentiation; growth arrest
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