A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

Although dispensable in vitro, HIV Nef enables high-level viral replication in infected hosts by an as yet unexplained mechanism. Previously, we proposed that Nef functionally cooperates with the viral transactivator Tat by derepressing the viral promoter via a Nef-associated kinase complex (NAKC). Here, we demonstrate that hnRNP-K, a host factor thought to facilitate crosstalk between kinases and gene expression, interacts with Nef and, as part of NAKC, nucleates Nef-interacting kinases, including Lck, PKCdelta, and PI-3 kinase, leading to Lck and Erk1/2 activation. This strongly increased HIV transcription, which depended on Tat and the NF-kB motif in the viral promoter, but not on NF-kB activation. Depletion of hnRNP-K in a Jurkat model of HIV latency increased Erk1/2 activity and greatly augmented HIV reactivating stimuli. We conclude that hnRNP-K coordinates membrane signaling with transcriptional derepression through Erk1/2 and is targeted by HIV to enable Tat-mediated transcription.

The relationship between glutamate and γ-aminobutyric acid (GABA) levels in the living human brain and pain sensitivity is unknown. Combined glutamine/glutamate (Glx), as well as GABA levels can be measured in vivo with single-voxel proton magnetic resonance spectroscopy. In this cross-sectional study, we aimed at determining whether Glx and/or GABA levels in pain-related brain regions are associated with individual differences in pain sensitivity. Experimental heat, cold, and mechanical pain thresholds were obtained from 39 healthy, drug-free individuals (25 men) according to the quantitative sensory testing protocol and summarized into 1 composite measure of pain sensitivity. The Glx levels were measured using point-resolved spectroscopy at 3 T, within a network of pain-associated brain regions comprising the insula, the anterior cingulate cortex, the mid-cingulate cortex, the dorsolateral prefrontal cortex, and the thalamus. GABA levels were measured using GABA-edited spectroscopy (Mescher-Garwood point-resolved spectroscopy) within the insula, the anterior cingulate cortex, and the mid-cingulate cortex. Glx and/or GABA levels correlated positively across all brain regions. Gender, weekly alcohol consumption, and depressive symptoms were significantly associated with Glx and/or GABA levels. A linear regression analysis including all these factors indicated that Glx levels pooled across pain-related brain regions were positively associated with pain sensitivity, whereas no appreciable relationship with GABA was found. In sum, we show that the levels of the excitatory neurotransmitter glutamate and its precursor glutamine across pain-related brain regions are positively correlated with individual pain sensitivity. Future studies will have to determine whether our findings also apply to clinical populations.

The N-terminus of the human immunodeficiency virus (HIV) pathogenicity factor Nef associates with a protein complex (NAKC for Nef-associated kinase complex) that contains at least two kinases: the tyrosine kinase Lck and a serine kinase activity which was found to phosphorylate Lck and the Nef N-terminus. Here we show that this serine kinase activity is mediated by members of the novel Protein Kinase C (nPKC) subfamily, PKCdelta and theta. Association with the Nef N-terminus was sufficient to activate PKC leading to phosphorylation of Nef in vitro on a conserved serine residue at position 6. Mutation of serine 6 or coexpression of a transdominant negative PKC mutant significantly reduced Nef-stimulated HIV transcription and replication in resting PBMC. When analyzing the molecular mechanisms, we found that mutating serine 6 moderately affected myristoylation of Nef and its association with Pak2 activity, whereas CD4 downmodulation was not inhibited. More interestingly, this mutation abolished the typical perinuclear localization of Nef in T cells. We conclude that the activation of nPKCs by Nef is required to increase viral replication/infectivity and direct the subcellular localization of Nef.

The Nef protein of HIV-1 is a key promoter of disease progression, owing to its dramatic yet ill-defined impact on viral replication. Previously, we have shown that Nef enhances embryonic ectodermal development Tat-mediated transcription in a manner depending on Lck and the cytoplasmic sequestration of the transcriptional repressor embryonic ectodermal development. In this study, we report that Lck is activated by Nef and targets protein kinase C downstream, leading to the translocation of the kinase into membrane microdomains. Although microdomain-localized protein kinase C is thought to induce the transcription factor NFB, we unexpectedly failed to correlate Nef-induced signaling events with enhanced NFB activity. Instead, we observed an increase in ERK MAPK activity. We conclude that Nef-mediated signaling cooperates with Nef-induced derepression and supports HIV transcription through an ERK MAPK-dependent, but NFB-independent, pathway.

BackgroundSignaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses.ResultsHere, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.ConclusionsIn summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

Signals emanating from antigen receptors critically regulate immune cell activation, survival, and differentiation. Transmembrane adapter proteins (TRAPs), a group of molecules that organize signaling complexes at the plasma membrane, play a pivotal role in propagating and fine-tuning antigen receptor-mediated signaling. During the last years, it has been demonstrated that most of the TRAPs possess inhibitory functions, including linker for activation of T cells (LAT), the best characterized adapter that links the T-cell receptor (TCR) to Ca(2+) flux and mitogen-activated protein kinase activation. Indeed, it appears that LAT may assemble inhibitory complexes that trigger negative feedback loops, thus terminating T-cell activation. Additionally, recent data demonstrate that SIT [Src homology 2 domain-containing phosphatase 2 (SHP2)-interacting TRAP] fine-tunes TCR-mediated signaling events and negatively regulates T-cell development and homeostasis. The experimental evidence suggests that TRAPs play a crucial role also in establishing tolerance. In fact, loss of SIT, LAX, or NTAL (non-T cell activation linker)/linker for activation of B cells (LAB) resulted in the spontaneous development of autoimmune diseases. Moreover, we recently showed that in addition to the inhibition of Src-family kinases, PAG (phosphoprotein associated with glycosphingolipid-enriched domains) is also involved in the negative regulation of Ras activation. Collectively, these data demonstrate that TRAPs are important modulators of immune cell activation and function. Finally, it appears that TRAPs possess redundant yet not completely overlapping functions.