Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

Abstract

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70ACT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70ACT104 but not Rsk Activation of p7OACT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p7OACT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.The advent of selective inhibitors of phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) (1) has begun to clarify the role of this enzyme in cell signaling. Wortmannin (2) and/or the structurally unrelated LY294002 (3) inhibits mitogenesis (2, 4), receptor-activated secretory (2) and transport responses-e.g., glucose transport and GLUT4 translocation (3, 5)-and receptor regulation of intracellular enzymes-e.g., insulin regulation of adipocyte lipolysis (5) and activation of p70 S6 kinase (4, 6). As to the biochemical mechanisms by which PI 3-kinase participates in these responses, the ability of PI 3-kinase to regulate membrane dynamics is best characterized in yeast, where VPS34, the major PI 3-kinase in Saccharomyces cerevisiae, is an essential protein required for the sorting of polypeptides to the regulated or constitutive secretory pathway (7). The immediate downstream target and effector pathway for PI 3-kinase action in this system remains obscure.The p70 S6 kinase is a ubiquitous mitogen-activated Ser/Thr kinase (8) that is necessary for cells to enter S phase after mitogen stimulation (9, 10). p70 and its closest homologs, the Rsk enzymes, are coordinately activated within minutes after mitogen addition through Ser/Thr phosphorylation of the enzyme (9). Abundant evidence indicates that the Rsks are activated in a Ras-dependent way (11), entirely as a result of their phosphorylation by the mitogen-activated protein kinases, erkl and erk2 (12)(13)(14). In contrast, p70 requires multiple independent inputs for activation. One input is directed at a pseudosubstrate autoinhibitory (SKAIPS) domain in the p70 noncatalytic carboxyl-terminal tail (15,16). Concomitant with activation in situ, this segment undergoes Ser/Thr phosphorThe publication costs of this article were defrayed in part by page...

Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation

“…In keeping with this observation, the inhibition pattern observed in B cells was quite similar to that seen in 32D cells. Wortmannin partially inhibited the protection from apoptosis by IL-4 in both resting [22] and LPS-activated B cells with an inhibitory concentration (IC50 80 nM) consistant with its published affects on PI 3' kinase in cells [38], [41][42][43]. Similar results were obtained with LY294002, a competitive inhibitor of PI 3' kinase [22 and data not shown].…”

Specific TrkA survival signals interfere with different apoptotic pathways

“…However, recent studies using mutant TrkA receptors indicate that the activation of PI3-kinase depends in the main on phosphorylation of tyrosine Y490, the SHC binding site in human TrkA (Baxter et al, 1995). The PI3-kinase targets S6-kinase (Weng et al, 1995) and Akt/PKB (Burgering and Co er, 1995;Franke et al, 1995) Figure 2b, respectively). As controls for Akt/PKB activation we stimulated cells with EGF (100 ng/ml) or PDGF-AB (50 ng/ml); both of which bound to endogenous receptors in ®broblasts and activated Akt/PKB.…”

Critical Role of T-Loop and H-Motif Phosphorylation in the Regulation of S6 Kinase 1 by the Tuberous Sclerosis Complex

“…Subsequently, two sites conserved among kinases of the AGC superfamily of serine/threonine kinases are phosphorylated, namely the activation loop (T-loop) site at position Thr-229 and the hydrophobic motif (H-motif) site at position Thr-389. Mutation of Thr-229 to either alanine or glutamate abolishes kinase activity (13)(14)(15). However, whereas substitution of Thr-389 with alanine abolishes kinase activity (13,16,17), glutamate substitution of the H-motif increases basal kinase activity (13,16,17).…”

“…Regulation of S6K1 by the growth factor adequacy pathway involves activation of phosphatidylinositol 3-kinase (PI3K) and, thus, the generation of phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) in cellular membranes. S6K1 is activated by ectopic expression of PI3K (14) and is inhibited by the PI3K inhibitor wortmannin (12,14), indicating that PI3K is both necessary and sufficient for S6K1 activity.…”