Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast

Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and nu merous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in respon se to mitogens depends on the activation of a multiprotein complex formed o n the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our d ata indicate that at least two defects are responsible for the decreased c- fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the T CF, Elk-1. In nuclei isolated from serum stimulated senescent cells the act ivating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the member s of the extracellular-regulated kinase (ERK) family was strikingly diminis hed and correlated with a decrease in the abundance of activated ERK protei ns. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consisten tly exhibited higher ERK protein abundance. Thus, the proportion of phospho rylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphoryla ted (active) MEK. These data indicate that in senescent cells there is a ge neral dysregulation in the ERK signaling pathway, which results in the accu mulation of inactive ERK molecules, decreased abundance of active ERK in th e nucleus of senescent cells, and subsequent lack of activation of the tran scription factor TCF Elk-1. These impairments, together with the impaired D NA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dep endent upon this pathway. (C) 2001 Academic Press.