Identification and characterization of a mitogen-activated S6 kinase.

Abstract

Treatment of Swiss mouse 3T3 cells with epidermal growth factor, orthovanadate, or serum results in the activation of a kinase that phosphorylates protein S6 of the 40S ribosomal subunit in vitro. This kinase is eluted as a single peak of activity from either a Mono Q anion-exchange column at 0.34 M NaCl or a Mono S cation-exchange column at 0.20 M NaCl. Treatment of the peak fraction from the Mono S column with phosphatase 2A completely abolishes the activity of the enzyme. The kinase appears to be distinct from protein kinase C, cAMP-dependent protein kinase, and two proteaseactivated kinases, PAK H and H4P. The kinase has been purified to apparent homogeneity and migrates as a single band at Mr 70,000 in NaDodSOn/polyacrylamide gels. The kinase exhibits the ability to autophosphorylate, and this activity directly parallels S6 phosphorylation activity on the final step of purification. In vitro, the kinase incorporates up to 5 mol of phosphate into S6, and the tryptic phosphopeptide maps obtained are equivalent to those from S6 phosphorylated in vivo. Most important, treatment of the purified kinase with phosphatase 2A results in complete inactivation of the enzyme, arguing that the activity of the kinase is directly controlled by phosphorylation.The activation of protein synthesis and subsequent cell growth in numerous biological systems is preceded by the multiple phosphorylation of S6, a protein of the 40S ribosomal subunit (1-6). In the case of quiescent animal cells stimulated to proliferate by specific mitogens, protein synthesis increases by as much as 3-fold and up to 5 mol of phosphate are incorporated per mol of S6 (7,8). The function of S6 phosphorylation in this system is not clearly understood; however, a central role in controlling the initiation of protein synthesis has been suggested by several lines of evidence (9-15).The possibility that protein phosphorylation cascades may be involved in the activation of cell growth has recently focused a great deal of attention on the kinase that phosphorylates S6 (16-24). Earlier we described an S6 kinase activity from Swiss mouse 3T3 cells that was stimulated up to 25-fold by serum, epidermal growth factor (EGF), or sodium orthovanadate (vanadate) (16,17). The S6 kinase activity in extracts prepared from cells treated with increasing concentrations of EGF was found to closely parallel the dose-response curve observed for S6 phosphorylation in the intact cell (16). In addition, the two-dimensional tryptic phosphopeptide maps derived from S6 phosphorylated in vitro and in vivo were equivalent (25). Together, these two findings suggested that the S6 kinase activity being followed in vitro was responsible for phosphorylating S6 in the intact cell.To recover full kinase activity from cell extracts, it was crucial to include phosphatase inhibitors such as glycerol 2-phosphate (16, 17). This finding suggested that the kinase might be regulated by phosphorylation and that the kinase inactivator in cell extracts was a phosphatase. Recently, we confirmed t...

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

“…Polyomavirus middle T antigens lacking tyrosine 315, a major site of phosphorylation by pp6Ocsr" in immune complexes, have impaired transforming potential (12,42 (2,24,32,37).…”

Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression

“…When activated by the convergence of mTOR-and PI3K-dependent signals, S6K1 functions as an in vivo kinase toward the 40S ribosomal protein S6 (Jeno et al, 1988), which has been suggested to increase the translational efficiency of a class of mRNA transcripts that contain a terminal oligopyrimidine (TOP) tract at their 5 0 -end (Jefferies et al, 1994(Jefferies et al, , 1997Terada et al, 1994). Many of these 5 0 -TOP mRNAs encode components of the translation machinery, such as ribosomal proteins and elongation factors (reviewed in Sonenberg and Gingras, 1998).…”

Inhibition of DNA Synthesis by a Farnesyltransferase Inhibitor Involves Inhibition of the p70s6k Pathway

“…One PI 3-kinase-dependent pathway required for mitogenic signaling involves p70 s6k (15,16). This enzyme was initially identified as a kinase capable of phosphorylating the ribosomal S6 protein in a growth factor-sensitive manner (17,18). Phosphorylation of S6 is thought to regulate the synthesis of proteins whose transcripts contain 5Ј-oligopyrimidine tracts.…”