Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

Abstract

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70' (Mr 70000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were asoated with p70" activation, the kinase was labeled to high specific activity with 32P1 in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensonal tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide ofMr 2400. Analysis of the p70"" protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vvo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p7O"". In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.Growth factors induce quiescent cells in culture to reenter the cell cycle, replicate their DNA, and divide through a complex array ofbiochemical responses (1). In most cases this process is initiated through specific ligand-activated receptor tyrosine kinases at the cell surface but is orchestrated intracellularly by a network of activated serine/threonine kinases (2). The mechanisms by which tyrosine kinases activate serine/ threonine kinases are still unclear, with the possible exception of protein kinase C (2, 3). One of the many early obligatory responses elicited by growth factors is the activation and maintenance of high rates of protein synthesis, which is required for reentry and transit through the G1 phase of the cell cycle (4, 5). This event is associated with increased phosphorylation of serine/threonine residues in a number of specific translational components, including 40S ribosomal protein S6 (6, 7). In vitro and in vivo studies have indicated that phosphorylation at five serine residues at the carboxyl terminus of S6 is required for the activation of protein synthesis (8-11). The kinase mediating this event was first detected in extracts of quiescent cells stimulated to proliferate by serum or epidermal growth factor (12, 13). Purification of this S6 kinase activity eventually revealed an enzyme of Mr 70,000, referred to as p70s6k (14,15), which is highly specific for ribosomal protein S6 (14), biphasically activated (16, 17), and selectively inactivated by type 2A phosphatase (18). The p70s"s phosphorylates four and possibly all five ofthe S6 sites observed in vivo (19), with recognition of the substrate absolutely dependent o...

Insulin restores differentiation of Ras-transformed C2C12 myoblasts by inducing NF-κB through an AKT/P70S6K/p38-MAPK pathway

“…We have investigated the contribution of these pathways to the di erentiation process by the use of chemical inhibitors. P70S6K was inhibited with the immunosuppressant rapamycin (Ferrari et al, 1992). To inhibit the a and b isoforms of p38-MAPK we used the pyridinyl imidazole compound PD169316 (PD*) (Kummer et al, 1997).…”

The Insulin-Induced Signalling Pathway Leading to S6 and Initiation Factor 4E Binding Protein 1 Phosphorylation Bifurcates at a Rapamycin-Sensitive Point Immediately Upstream of p70s6k

“…In parallel it has been demonstrated that the immunosuppressant rapamycin, a bacterial macrolide, negates mitogen-induced activation of p70 s6k by preventing the acute phosphorylation of a specific subset of sites, including T 229 , T 389 , S 404 , and S 411 (54). These sites were found to be flanked by large aromatic residues, with the exception of S 411 , which exhibits an S/TP motif, as do the remaining three unaffected phosphorylation sites (18,54). Of the rapamycin-sensitive sites, the principal site of rapamycin-induced dephosphorylation leading to p70 s6k inactivation was identified as T 389 , which resides in the linker region, coupling the catalytic and autoinhibitory domains (54).…”

The Activated Glucocorticoid Receptor Modulates Presumptive Autoregulation of Ribosomal Protein S6 Protein Kinase, p70 S6K

“…Furthermore, incubation of p70 S6K with synthetic peptides derived from this homologous region substantially hinders its activation (68, 69) demonstrating that this module, in the context of the full-length p70 S6K polypeptide, may serve an autoinhibitory function. Stimulation of cells in culture with growth factors initiates signals that lead to the hierarchical activation of p70 S6K, commencing with the (Ser/Thr)Pro-specific phosphorylation of a cluster of 4 -6 residues with the carboxyl terminus of the kinase (19). This battery of phosphorylations promotes a conformational change that relieves autoinhibition imposed by the carboxyl terminus and, in addition, exposes additional phosphorylation sites initially buried within interior of p70 S6K.…”

“…Two key observations support this premise. First, the primary amino acid sequence surrounding the cluster of p70 S6K carboxyl-terminal phosphorylation sites exhibits strong homology to the region within its substrate, S6, that it phosphorylates (19,68); hence, the designation "pseudosubstrate" domain. Furthermore, incubation of p70 S6K with synthetic peptides derived from this homologous region substantially hinders its activation (68, 69) demonstrating that this module, in the context of the full-length p70 S6K polypeptide, may serve an autoinhibitory function.…”

“…1A). These phosphorylations occur in a hierarchical manner commencing with phosphorylation of a cluster of at least four carboxyl-terminal sites localized within the presumptive autoinhibitory pseudosubstrate domain (19). These phosphorylation events have been suggested to transform the conformation of the protein so as to disrupt a putative interaction between the autoinhibitory region and the substrate recognition pocket.…”