Paul C. Sternweis

A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits

Abstract Phosphoinositide 3 kinase (PI3K) is a key signaling enzyme implicated in receptor-stimulated mitogenesis, oxidative bursting in neutrophils, membrane ruffling, and glucose uptake. A PI3K has already been purified, cloned, and shown to be regulated by receptors that act via tyrosine kinase-dependent regulatory mechanisms. We report that an immunologically, pharmacologically, and chromatographically distinct form of PI3K activity present in neutrophils and U937 cells is specifically activated by G protein βγ subunits. This data suggests PI3Ks conform to the paradigm set by receptor regulation of phosphoinositidase Cs: different receptor transduction systems specifically regulate dedicated isoforms of effector protein.

Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins.

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1,2,3,4,5). GLAST and GLT-1 are localized primarily in astrocytes, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9,10,11) and EAAT5 (ref. 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4—the glutamate transporter expressed predominately in the cerebellum—or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

A global analysis of cross-talk in a mammalian cellular signalling network

Cellular information processing requires the coordinated activity of a large network of intracellular signalling pathways. Cross-talk between pathways provides for complex non-linear responses to combinations of stimuli, but little is known about the density of these interactions in any specific cell. Here, we have analysed a large-scale survey of pathway interactions carried out by the Alliance for Cellular Signalling (AfCS) in RAW 264.7 macrophages. Twenty-two receptor-specific ligands were studied, both alone and in all pairwise combinations, for Ca2+ mobilization, cAMP synthesis, phosphorylation of many signalling proteins and for cytokine production. A large number of non-additive interactions are evident that are consistent with known mechanisms of cross-talk between pathways, but many novel interactions are also revealed. A global analysis of cross-talk suggests that many external stimuli converge on a relatively small number of interaction mechanisms to provide for context-dependent signalling.

Regulation of phospholipase C by G proteins

Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the Gq subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the Gq subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.

Differential G protein—mediated coupling of neurotransmitter receptors to Ca2+ channels in rat dorsal root ganglion neurons in vitro

The peptides neuropeptide Y (NPY) and bradykinin (BK) both inhibited Ca2+ currents in rat dorsal root ganglion neurons (DRG) in vitro. The effects of both peptides were completely blocked by treatment of cells with pertussis toxin. Based on antigenic determinants, DRG cells contained at least two pertussis toxin substrates, alpha o (Mr, 39 kd) and alpha i2 (Mr, 40 kd). We examined the ability of three purified bovine alpha subunits (identified with antibodies as alpha o, alpha i1, and alpha i2) to reconstitute the inhibitory effects of NPY and BK. Reconstitution of NPY effects occurred according to the potency series alpha o greater than alpha i1 much greater than alpha i2. However, in the case of BK all three G proteins were approximately equally effective. Whereas complete reconstitution of NPY effects could be obtained with alpha o, no single alpha subunit produced complete reconstitution of BK. Combinations of alpha o and alpha i2, however, were able to completely reconstitute the effects of BK. Thus several G proteins can effect the regulation of Ca2+ channels in these cells. However, neurotransmitters may be selective in the G proteins or combinations of G proteins utilized to achieve this regulation.

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Regulation of intracellular cyclic adenosine 3 ′,5 ′-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5 ′-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

Suppression of LPS-Induced TNF-α Production in Macrophages by cAMP Is Mediated by PKA-AKAP95-p105

Distinct anchoring proteins enable cAMP signaling to selectively modulate macrophage responses to pathogens. Specific Scaffolds Macrophages are innate immune cells that mediate early responses to infection by sensing microbial products through Toll-like receptors (TLRs) and producing proinflammatory compounds, such as tumor necrosis factor–α (TNF-α). A modulator of this proinflammatory response is prostaglandin E2 (PGE2), which activates G protein–coupled receptors that couple to Gαs, leading to the production of cyclic adenosine monophosphate (cAMP). As well as inhibiting the production of TNF-α by macrophages in response to the TLR4 agonist LPS, PGE2 and cAMP also stimulate the production of the anti-inflammatory cytokines interleukin-10 (IL-10) and granulocyte colony-stimulating factor (G-CSF) (see the Perspective by Peters-Golden). Wall et al. found that the pleiotropic effects of PGE2 and cAMP on LPS-stimulated cytokine production depended on the fate of cAMP-dependent protein kinase (PKA). Selective binding of activated PKA to different scaffold proteins known as A kinase–anchoring proteins (AKAPs) resulted in differential effects on the expression of genes encoding cytokines. In particular, cAMP-dependent inhibition of TNF-α expression involved phosphorylation of the NF-κB transcription factor p105 by PKA bound to AKAP95, which inhibited the nuclear translocation of the transcription factor, whereas the effect of PKA on the enhancement of G-CSF expression was mediated by another AKAP; the effect of PKA on IL-10 expression was AKAP-independent. Together, these data uncover crosstalk between TLR4 and cAMP signaling pathways that depend on the differential localization of PKA by different scaffold proteins, which could have implications for anti-inflammatory therapies. 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 E2, 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.

Overview of the Alliance for Cellular Signaling

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

The active role of βγ in signal transduction

Abstract Many receptors that sense the environment effect intracellular regulation through stimulation of heterotrimeric G proteins and the consequences thereof. While prominence was originally given to the α-subunits of G proteins as the pathway for downstream regulation, very active roles for the βγ-subunits have emerged in the past year. Recent experiments highlight the versatility of βγ-subunits in these regulatory pathways, but also emphasize some fundamental questions that remain.

The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate phosphatase activities in Saccharomyces cerevisiae

Two genes in Saccharomyces cerevisiae, LPP1 and DPP1, with homology to a mammalian phosphatidic acid (PA) phosphatase were identified and disrupted. Neither single nor combined deletions resulted in growth or secretion phenotypes. As observed previously (Toke, D. A., Bennett, W. L., Dillon, D. A., Wu, W.-I., Chen, X., Ostrander, D. B., Oshiro, J., Cremesti, A., Voelker, D. R., Fischl, A. S., and Carman, G. M. (1998) J. Biol. Chem. 273, 3278–3284; Toke, D. A., Bennett, W. L., Oshiro, J., Wu, W.-I., Voelker, D. R., and Carman, G. M. (1998) J. Biol. Chem. 273, 14331–14338), the disruption of DPP1 and LPP1 produced profound losses of Mg2+-independent PA phosphatase activity. The coincident attenuation of hydrolytic activity against diacylglycerol pyrophosphate prompted an examination of the effects of these disruptions on hydrolysis of isoprenoid pyrophosphates. Disruption of either LPP1 or DPP1 caused respective decreases of about 25 and 75% in Mg2+-independent hydrolysis of several isoprenoid phosphates by particulate fractions isolated from these cells. The particulate and cytosolic fractions from the double disruption (lpp1Δ dpp1Δ) showed essentially complete loss of Mg2+-independent hydrolytic activity toward dolichyl phosphate (dolichyl-P), dolichyl pyrophosphate (dolichyl-P-P), farnesyl pyrophosphate (farnesyl-P-P), and geranylgeranyl pyrophosphate (geranylgeranyl-P-P). However, a modest Mg2+-stimulated activity toward PA and dolichyl-P was retained in cytosol fromlpp1Δ dpp1Δ cells. The action of Dpp1p on isoprenyl pyrophosphates was confirmed by characterization of the hydrolysis of geranylgeranyl-P-P by the purified protein. These results indicate that LPP1 and DPP1 account for most of the hydrolytic activities toward dolichyl-P-P, dolichyl-P, farnesyl-P-P, and geranylgeranyl-P-P but also suggest that yeast contain other enzymes capable of dephosphorylating these essential isoprenoid intermediates.