p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitorypseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKC as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKC mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKC mutant (myristoylated PKC [myr-PKC ]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a Cterminal truncation mutant of p70S6K was also enhanced by myr-PKC . Moreover, we have found that p70S6K can associate with both PDK-1 and PKC in vivo in a growth factor-independent manner, while PDK-1 and PKC can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.

Results confirm the recognized importance of age and upper and lower extremity strengths for walking after a SCL. They also highlight the role of 2 other factors, i.e., balance and spasticity, seldom considered as thoroughly in SCL.

Phosphorylation of the highly conserved hydrophobic motif site in AGC kinases is necessary for phosphotransferase activity. Phosphorylation of this motif (FLGFT389Y) in p70 S6 kinase (S6K1) is both rapamycinand wortmannin-sensitive, suggesting a role for both mammalian target of rapamycin-and phosphatidylinositol 3-kinase-dependent pathways. We report here that co-expression of phosphoinositide-dependent kinase-1 (PDK1) and the phosphatidylinositol 3-kinaseregulated atypical protein kinase C cooperate to increase both phosphorylation of the hydrophobic motif site Thr 389 , as well as the activation loop site Thr 229 . Interestingly, although PDK1 alone can promote an increase in Thr 389 phosphorylation in both wild type S6K1 and a kinase-inactive mutant of S6K1, the cooperative effect between PDK1 and protein kinase C required S6K1 activity. Furthermore, Akt, another phosphatidylinositol 3-kinase effector and regulator of S6K1, also increased Thr 389 phosphorylation in a S6K1 activity-dependent manner. Consistent with this, epidermal growth factor-induced Thr 389 phosphorylation in wild type S6K1 persisted for up to 120 min, whereas kinaseinactive mutants of S6K1 displayed only a reduced and transient increase in Thr 389 phosphorylation. We conclude that S6K1 activity is required for maximal Thr 389 phosphorylation by mitogens and by multiple phosphatidylinositol 3-kinase-dependent inputs including PDK1, PKC, and Akt, and we propose that autophosphorylation is an important regulatory mechanism for phosphorylation of the hydrophobic motif Thr 389 site in S6K1.

Ribosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitro and is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase C but that S6K2 is more sensitive to basal activation by myristoylated protein kinase C than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases.The 70-kDa ribosomal S6 kinase 1 (S6K1) 1 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in response to mitogen stimulation (1). S6 phosphorylation up-regulates translation of mRNAs with 5Ј-terminal oligopyrimidine tracts, many of which encode ribosomal proteins and translation elongation factors (2). S6K1 activation thus up-regulates ribosome biosynthesis and enhances the translational capacity of the cell.Deletion of S6K1 in Drosophila and mice has implicated S6K1 in regulation of cell size. The Drosophila knockout has a high incidence of embryonic lethality, but surviving flies exhibit a marked reduction in size that is cell autonomous (3). Mice lacking S6K1 through targeted disruption also exhibit a small animal phenotype (4). Interestingly, S6 phosphorylation and 5Ј-terminal oligopyrimidine tract mRNA translation were found to be normal in fibroblasts derived from mice lacking S6K1, suggesting a compensatory mechanism for these S6K1 functions. Our lab and others have recently identified S6K2, a homolog of S6K1 that phosphorylates S6 in vitro (4 -7). Elevated S6K2 mRNA levels have been reported in the S6K1 knockout mice (4). Drosophila are thought to express only S6K1, which may account for the more severe S6K1 knockout phenotype in flies. S6K2 is a good candidate kinase that may supply some but not all of the functions of S6K1 in the knockout mouse, because the small animal phenotype persists despite the presence of S6K2, S6 phosphorylation, and 5Ј-terminal oligopyrimidine tract mRNA translation.There are two is...