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 G s -dependent receptors for isoproterenol and prostaglandin E 2 . Whereas two ligands, uridine 5-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G q /calcium and G i , 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 G s -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 S1P 2 receptor and the heterotrimeric G 13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.Cyclic adenosine 3Ј,5Ј-monophosphate (cAMP), a ubiquitous second messenger, mediates a wide range of cellular functions including cell metabolism (1), cell proliferation and differentiation (1), immune responses (2, 3), memory formation (4), and cardiac contractility (5). Canonically, the concentration of intracellular cAMP is regulated by two distinct families of enzymes. The transmembrane adenylyl cyclases (ACs) 3 synthesize cAMP from adenosine triphosphate (6, 7), whereas the cAMP-specific phosphodiesterases metabolize cAMP to biologically inactive adenosine 5Ј-monophosphate (8, 9). ACs are primarily activated by G␣ s but their activities can also be differentially regulated by G␣ i , G␤␥, or Ca 2ϩ (10, 11). The activities of various phosphodiesterases can be regulated by protein kinase A (PKA), extracellular-regulated kinase (ERK), phosphoinositide 3-kinase, and the concentration of cAMP itself (12-16). Thus integration of signaling by stimuli that can regulate the intracellular concentration of cAMP will depend strongly on the various pathways and the subtypes of ACs and phosphodiesterases expressed in individual cells at any given time.Assessment of the regulation of intracellular cAMP in vivo has only become possible recently. Zaccolo et al. (17) first described a FRET sensor for cAMP based on the cAMP binding domain of PKA. Subsequently, several reports have described FRET sensors for cAMP based on binding of the nucleotide to the Epac proteins (18 -21). While these FRET sensors have been effective for measuring changes and localization of cAMP in single cells, measurements are tedious. Furthermore, the requirement for excitation of donor molecules pro...

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.

Both the ␤-catenin and the nuclear factor B (NF-B) proteins are important regulators of gene expression and cellular proliferation. Two kinases, IKK␣ and IKK␤, are critical activators of the NF-B pathway. Here we present evidence that these kinases are also important in the regulation of ␤-catenin function. IKK␣-and IKK␤-deficient mouse embryo fibroblasts exhibited different patterns of ␤-catenin cellular localization. IKK␤ decreases ␤-catenin-dependent transcriptional activation, while IKK␣ increases ␤-catenin-dependent transcriptional activity. IKK␣ and IKK␤ interact with and phosphorylate ␤-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of ␤-catenin with IKK␣ and IKK␤ may in part be responsible for regulating ␤-catenin protein levels and cellular localization and integrating signaling events between the NF-B and Wingless pathways.␤-Catenin, the mammalian homologue of the Drosophila armadillo protein, is a ubiquitously expressed protein that has at least two distinct roles in the cell. First, it participates in cell-cell adhesion by mediating the association of E-cadherin with the cytoskeleton (1, 2). Second, it is a critical downstream component of the Wnt 1 or Wingless signal transduction pathway (3-5). The Wnt family of secretory glycoproteins plays an important role in embryonic development, in the induction of cell polarity, and in the determination of cell fate. Deregulation of Wnt signaling disrupts axis formation in embryos (5-8) and is associated with multiple human malignancies (9).The current model of Wnt signaling indicates that the binding of the Wnt proteins to their receptor, frizzled, stabilizes ␤-catenin by inhibiting the activity of a serine/threonine kinase glycogen synthase kinase-3 or GSK-3␤ (9). GSK-3␤ is associated with ␤-catenin in a multiprotein complex that includes the adenomatous polyposis coli tumor suppressor protein (APC), axin or conductin, protein phosphatase 2A, and dishevelled. GSK-3␤ phosphorylation of residues in the amino terminus of ␤-catenin results in APC-mediated ␤-catenin degradation via the ubiquitin-proteosome pathway (10, 11). Increased levels of ␤-catenin are frequently found in colon cancer due to mutations in either the APC gene (12)(13)(14) or at residues in the amino terminus of ␤-catenin that are phosphorylated by . In the nucleus, ␤-catenin forms a complex with members of the T-cell factor (TCF)/lymphocyte-enhancer factor (LEF) family and activates gene expression of a variety of target genes (18 -23) including c-myc (24) and cyclin D1 (25, 26).NF-B comprises a family of transcription factors which are critical in activating the expression of genes involved in the immune and inflammatory response and in the regulation of cellular apoptosis (27, 28). NF-B is sequestered in the cytoplasm by a family of inhibitory proteins known as IB. Upon stimulation of this pathway by a variety of agents including IL-1 and TNF␣, the kinases IKK␣ and IKK␤ (29 -33) in conjunction with the scaffold protein IKK␥/NEMO (34 -36) l...

Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/ arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.Dynamin is a GTPase required for membrane internalization during synaptic vesicle recycling and receptor-mediated endocytosis (for recent reviews, see Refs. 1-5). GTP hydrolysis is necessary for the cellular functioning of dynamin since overexpression of inactive dynamin mutants elicits a dominant inhibitory effect on host cell endocytosis (6, 7). Therefore, the regulation of this enzymatic activity has been under intense investigation. GTPase activity is tightly coupled to dynamin self-assembly, which can occur in the absence of other molecules (8 -10), but is facilitated by multivalent surfaces provided by microtubules (11-15) or anionic liposomes (15). Specific activity is also increased by phosphorylation (16) and by interaction with several SH3 domain-containing proteins (17, 18). The mechanisms by which these interactions regulate dynamin GTPase activity are poorly understood.Until recently it was believed that all dynamin activators interact with a carboxyl-terminal domain of approximately 100 residues designated PRD 1 for its high content of prolines (P) and arginines (R). Negatively charged molecules, such as microtubules and phosphatidylserine-containing liposomes, bind to the PRD via ionic interactions which are disrupted at physiological ionic strength (14). SH3 domain-containing proteins, including Grb2 and amphiphysin, bind tightly to specific proline-rich motifs located in the .Another potential site for dynamin interactions is the pleckstrin homology (PH) domain, a module found in numerous signa...

The actin cytoskeleton of nonmuscle cells undergoes extensive remodeling during agonist stimulation. Lamellipodial extension is initiated by uncapping of actin nuclei at the cortical cytoplasm to allow filament elongation. Many actin filament capping proteins are regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which is hydrolyzed by phospholipase C. It is hypothesized that PIP 2 dissociates capping proteins from filament ends to promote actin assembly. However, since actin polymerization often occurs at a time when PIP 2 concentration is decreased rather than increased, capping protein interactions with PIP 2 may not be regulated solely by the bulk PIP 2 concentration. We present evidence that PIP 2 binding to the gelsolin family of capping proteins is enhanced by Ca 2؉. Binding was examined by equilibrium and nonequilibrium gel filtration and by monitoring intrinsic tryptophan fluorescence. Gelsolin and CapG affinity for PIP 2 were increased 8-and 4-fold, respectively, by M Ca 2؉, and the Ca 2؉ requirement was reduced by lowering the pH from 7.5 to 7.0. Studies with the NH 2 -and COOH-terminal halves of gelsolin showed that PIP 2 binding occurred primarily at the NH 2 -terminal half, and Ca 2؉ exposed its PIP 2 binding sites through a change in the COOH-terminal half. Mild acidification promotes PIP 2 binding by directly affecting the NH 2 -terminal sites. Our findings can explain increased PIP 2 -induced uncapping even as the PIP 2 concentration drops during cell activation. The change in gelsolin family PIP 2 binding affinity during cell activation can impact divergent PIP 2 -dependent processes by altering PIP 2 availability. Cross-talk between these proteins provides a multilayered mechanism for positive and negative modulation of signal transduction from the plasma membrane to the cytoskeleton.Phosphoinositides are important in signal transduction, both as precursors to signaling molecules and as physical anchors and regulators of proteins (1, 2). Among these, the D4 phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 1 has been implicated as a potential mediator of actin cytoskeletal rearrangements (3, 4). PIP 2 modulates many actin regulatory proteins. These include the following: actin severing and/or capping proteins (gelsolin (5), CapG (6), and capping protein (also known as Cap Z) (7)), monomer-binding proteins (profilin (8) and cofilin (9)), and other actin-binding proteins (␣-actinin (10) and vinculin (11)). It has been hypothesized that PIP 2 induces explosive actin assembly by dissociating capping proteins from filament ends and releasing actin monomers from actin-sequestering proteins (3, 7, 12). The involvement of PIP 2 in actin polymerization is supported by recent experiments that show that Rac1 and RhoA, monomeric GTPases of the Rho family that have well defined effects on the cytoskeleton (13), stimulate the synthesis of PIP 2 (14 -16). Furthermore, manipulations that alter the availability of PIP 2 in cells have profound effects on agonist and/or Rac1-induced fil...

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ during agonist stimulation. A physiologically relevant rise in Ca2+ increases their affinity for PIP2 and can promote significant interactions with PIP2 in activated cells. This may impact divergent PIP2- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase Cγ (PLCγ) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP2 in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLCβ. Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLCβ, and only gelsolin domains containing PIP2-binding sites were effective. Inhibition was mitigated by increasing PIP2 concentration in a manner consistent with competition between gelsolin and PLCβ for PIP2. Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLCγ, although they inhibited PLCγ less than PLCβ. Our findings indicate that as PIP2 level and availability change during signaling, cross talk between PIP2-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.

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Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G proteincoupled receptors (GPCRs) in raising intracellular free Ca 2؉ . To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca 2؉ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was G␣ i -dependent, whereas the UDP, PAF, and LPA responses were G␣ q -dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca 2؉ from intracellular stores as well as sustained Ca 2؉ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) ␤. Macrophages expressed transcripts for three PLC␤ isoforms (PLC␤2, PLC␤3, and PLC␤4), but GPCR ligands selectively used these isoforms in Ca 2؉ signaling. C5a predominantly used PLC␤3, whereas UDP used PLC␤3 but also PLC␤4. Neither ligand required PLC␤2. Synergy between C5a and UDP likewise depended primarily on PLC␤3. Importantly, the Ca 2؉ signaling deficiency observed in PLC␤3-deficient BMDM was reversed by re-constitution with PLC␤3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca 2؉ , PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLC␤3 may thus provide a selective target for inhibiting Ca 2؉