Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth fa ctor stimulation of quiescent fibroblasts and inhibition of this enzyme res ults in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors , leading to an increase in protein synthesis. We have examined the activat ion of S6K1 in human fibroblasts following mitogen stimulation. In early pa ssage fibroblasts S6K1 is activated following serum stimulation as evidence d by increased kinase activity and site-specific phosphorylation. In contra st, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 ( Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoret ic mobility. Inhibitor studies indicate that this phosphorylation is depend ent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPR pathw ay. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 prot ein. In early passage fibroblasts the ribosomal S6 protein is phosphorylate d upon serum stimulation while the phosphorylation of the ribosomal S6 prot ein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative se nescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation. (C) 2000 Academic Press.