The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E 2 , that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-α, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor κB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates.
Transcription factors of the Forkhead Box, class O (FOXO) family promote cell-cycle arrest and/or apoptosis in a variety of cell types. Mitogenic stimuli inactivate FOXO function by way of an evolutionarily conserved pathway involving the activation of phosphoinositide 3-kinase (PI3K) and its downstream effector, Akt. Although PI3K activation is required for B-lymphocyte proliferation, it is not known whether PI3K-dependent inactivation of FOXO proteins is important for cell-cycle progression and survival of these cells. Here, we show that B-cell receptor (BCR) engagement triggers PI3K-dependent phosphorylation and nuclear export of FOXO1. Furthermore, forced expression of PI3K-independent variants of FOXO1 or FOXO3a in activated B cells induces partial arrest in G1 phase of the cell cycle and increases apoptosis. These findings establish that FOXO inactivation is a functionally important consequence of PI3K signaling in primary B cells.
Gamma interferon (IFN-␥) induces both tyrosine and serine phosphorylation of Stat1. Stat1 serine phosphorylation is required for maximal transcriptional activity of Stat1. In this report, we present evidence that Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed time course than tyrosine phosphorylation. The occurrence of serine phosphorylation without tyrosine phosphorylation suggests that serine phosphorylation takes place in the cytoplasm. Experiments performed with cells expressing either dominant-negative or constitutively active Ras protein indicated that the Ras-mitogenactivated protein kinase pathway is probably not involved in IFN-␥-induced Stat1 serine phosphorylation. Finally, a kinase capable of correct Stat1 serine phosphorylation was detected in partially purified cytoplasmic extracts from both IFN-␥-treated and untreated cells.Polypeptide signalling that changes gene expression often involves the STAT proteins (16). These latent cytoplasmic transcription factors become activated by phosphorylation of tyrosine residues, catalyzed either by receptor-associated Janus (Jak) kinases or by receptor tyrosine kinases. The STAT molecules then dimerize by reciprocal phosphotyrosine-SH2 interactions and enter the cell nucleus to effect transcriptional changes. It has been amply demonstrated that transcriptional activation is further amplified when the serine residues of STAT proteins are phosphorylated (22). A single serine in Stat3 and Stat1, residue 727 in both cases, appears to be the major, if not the only, serine kinase target site (21). In addition to these studies, a number of other findings suggestive of serine phosphorylation of STATs that are also tyrosine phosphorylated have been described. For example, Stat3 and Stat5 show a ligand-dependent, slower migration that is also time dependent and is inhibited by the serine kinase inhibitor H7 (2,3,24).The signalling pathway(s) involved in serine phosphorylation of the STATs is not known at present, although we do show here that Jak2 is required for STAT serine phosphorylation in response to gamma interferon (IFN-␥). In this study, we explored the time course and cellular locus of serine and tyrosine phosphorylation and the potential interdependence of the two. In addition, we examined the nature of the serine phosphorylation pathways, demonstrating the lack of evidence for participation of several prominent serine kinases. However, we did detect a kinase capable of catalyzing phosphorylation of Stat1, predominantly on residue 727. MATERIALS AND METHODSCell culture. U3A cells (13,14), ␥2A cells (12), and their derived cell lines were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% cosmic serum (HyClone Laboratories Inc.). NIH 3T3 cells and cells stably transfected with either dominant-negative Ras (dnRas) or constitutively active Ras (CARas) were grown in DMEM supple...
Although numerous strategies are now available to generate rudimentary forms of synthetic cell-like entities, minimal progress has been made in the sustained excitation of artificial protocells under non-equilibrium conditions. Here we demonstrate that the electric field energization of coacervate microdroplets comprising polylysine and short single strands of DNA generates membrane-free protocells with complex, dynamical behaviours. By confining the droplets within a microfluidic channel and applying a range of electric field strengths, we produce protocells that exhibit repetitive cycles of vacuolarization, dynamical fluctuations in size and shape, chaotic growth and fusion, spontaneous ejection and sequestration of matter, directional capture of solute molecules, and pulsed enhancement of enzyme cascade reactions. Our results highlight new opportunities for the study of non-equilibrium phenomena in synthetic protocells, provide a strategy for inducing complex behaviour in electrostatically assembled soft matter microsystems and illustrate how dynamical properties can be activated and sustained in microcompartmentalized media.
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