Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21 Sdi1/Cip1/Waf1 *

Expression of p21 Sdi1 downstream of p53 is essential for induction of cellular senescence, although cancer cell senescence can also occur in the p53 null condition. We report herein that senescence-associated phosphorylated extracellular signal-regulated protein kinases 1 and 2 (SA-pErk1/2) enhanced p21 Sdi1 transcription by phosphorylating Sp1 on Ser 59 downstream of protein kinase C (PKC) α. Reactive oxygen species (ROS), which was increased in cellular senescence, significantly activated both PKCα and PKCβI. However, PKCα, but not PKCβI, regulated ROS generation and cell proliferation in senescent cells along with activation of cdk2, proven by siRNAs. PKCα-siRNA also reduced SA-pErk1/2 expression in old human diploid fibroblast cells, accompanied with changes of senescence phenotypes to young cell-like. Regulation of SA-pErk1/2 was also confirmed by using catalytically active PKCα and its DN-mutant construct. These findings strongly suggest a new pathway to regulate senescence phenotypes by ROS via Sp1 phosphorylation between PKCα and SA-pErk1/2: employing GST-Sp1 mutants and MEK inhibitor analyses, we found that SA-pErk1/2 regulated Sp1 phosphorylation on the Ser 59 residue in vivo, but not threonine, in cellular senescence, which regulated transcription of p21 Sdi1 expression. In summary, PKCα, which was activated in senescent cells by ROS strongly activated Erk1/2, and the SA-pErk1/2 in turn phosphorylated Sp1 on Ser 59. Sp1-enhanced transcription of p21 Sdi1 resulted in regulation of cellular senescence in primary human diploid fibroblast cells.