The activation of p70 s6k is accompanied by a complex series of phosphorylation events. In this review we propose a model of activation which divides p70 s6k into four functional modules that cooperate in leading to full enzyme activity. In the light of the model, we suggest how candidate effectors of p70 s8k activation might function by directing the phosphorylation of specific sites.© 1997 Federation of European Biochemical Societies.Key words: Modular phosphorylation; p70 s6k1. The significance of p70 /p85 in cell cycle progressionThe discovery that mitogen-induced metabolic processes maybe regulated by serial phosphorylation and dephosphorylation events has captivated the attention of signal transduction scholars for over a decade [1]. The application of this finding has motivated workers to purify, clone and crystallise many of the proteins responsible for mediating specific mitogenic events. The goal of this laboratory has been to unravel the complex series of signalling events which lead to, and from, the activation of the p70/p85 isoforms of S6 kinase, p70 s6k /p85 s6k [2][3][4]. The p70 sfik is recognised as the kinase which regulates the multiple phosphorylation of 40S ribosomal protein S6 in vivo, distinct from the p21 ras -mediated p9Qrsk r5] inhibition of mitogen-induced p70 s6k /p85 s6k activation in vivo with either neutralising antibodies [6,7] or by treatment with the immunosuppressant rapamycin [8-10] severely compromises the ability of the cell to progress through the Gl phase of the cell cycle. The importance of these observations is emphasised by the critical role p70 s6k plays, presumably through S6 phosphorylation, in regulating the translation of a class of mRNA transcripts which contain an oligopyrimidine tract at their transcriptional start site [4]. This class of mRNAs encode for many of the components of the protein synthetic apparatus and can represent up to 20% of the total mRNA in the cell [11]. Failure to recruit these messages into polysomes suppresses the biogenesis of translational machinery required for cell cycle progression [12].The p70 s6k /p85 s6k isoforms are generated from alternative translation start sites on the same transcript (Y. Chen and S. Kozma, unpublished). The two isoforms are coordinately regulated and only differ by a 23 amino acid N-terminal extension which constitutively targets p85 s6k to the nucleus [7]. Whilst the mechanism by which p85 s8k mediates cell cycle progression is, as yet, unresolved, it is noteworthy that S6 is *Corresponding author. Fax: (41) (61) 697 6681. E-mail: gthomas@fmi.ch present in a free form in the nucleus and becomes phosphorylated at the same residues in response to mitogens as its cytoplasmic counterpart [13]. As the role of S6 in the cytoplasm and nucleus has been recently discussed [4,14], we will concentrate here on the upstream signalling elements and the modular mechanisms which culminate in p70 s6k activation. The putative signalling components that elevate p70 s6k activityThe observation that p70 s6k activity c...

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.

In this study, we investigated the effect of fiber and pore size of an electrospun scaffold on the polarization of mouse bone marrow-derived macrophages (BMMΦs) towards regenerative (M2) or inflammatory (M1) phenotypes. BMMΦs were seeded on Polydioxanone (PDO) scaffolds electrospun from varying polymer concentrations (60, 100, and 140 mg/ml). Higher polymer concentrations yielded larger diameter fibers with larger pore sizes and porosity. BMMΦ cultured on these scaffolds showed a correlation between increasing fiber/pore size and increased expression of the M2 marker Arginase 1 (Arg1), along with decreased expression of the M1 marker inducible nitric oxide synthase (iNOS). Secretion of the angiogenic cytokines VEGF, TGF-β1 and bFGF was higher among cultures employing larger fiber/pore size scaffolds (140 mg/ml). Using a 3D in vitro angiogenesis bead assay, we have demonstrated that the M2-like profile of BMMΦ induced by the 140 mg/ml is functional. Furthermore, our results show that the pore size of a scaffold is a more critical regulator of the BMMΦ polarization compared to the fiber diameter. The study also shows a potential role for MyD88 in regulating M1 BMMΦ signaling on the large vs. small fiber/pore size PDO scaffold. These data are instructive for the rationale design of implantable prosthetics designed to promote in situ regeneration.

Mitogen-induced activation of p70s6k is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85 s6k isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino-and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.

During flowering, primordia on the flanks of the shoot apical meristem are specified to form flowers instead of leaves. Like many plants, Arabidopsis thaliana integrates environmental and endogenous signals to control the timing of reproduction. To study the underlying regulatory logic of the floral transition, we used a combination of modeling and experiments to define a core gene regulatory network. We show that FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) act through FD and FD PARALOG to regulate the transition. The major floral meristem identity gene LEAFY (LFY) directly activates FD, creating a positive feedback loop. This network predicts flowering behavior for different genotypes and displays key properties of the floral transition, such as signal integration and irreversibility. Furthermore, modeling suggests that the control of TFL1 is important to flexibly counterbalance incoming FT signals, allowing a pool of undifferentiated cells to be maintained despite strong differentiation signals in nearby cells. This regulatory system requires TFL1 expression to rise in proportion to the strength of the floral inductive signal. In this network, low initial levels of LFY or TFL1 expression are sufficient to tip the system into either a stable flowering or vegetative state upon floral induction.