A chemoattractant-mediated Gi-coupled pathway activates adenylyl cyclase in human neutrophils

Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to Galpha(s)-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical Galpha(s)-meditated pathway.     

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Phosphoinositide 3-kinase controls early and late events in mammalian cell division

Phosphoinositide 3-kinase (PI3K) plays a crucial role in triggering cell division. To initiate this process, PI3K induces two distinct routes, of which one promotes cell growth and the other regulates cyclin-dependent kinases. Fine-tuned PI3K regulation is also required for later cell cycle phases. Here, we review the multiple points at which PI3K controls cell division and discuss its impact on human cancer.     

The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.     

Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.     

Phosphoinositide 3-kinase signaling in the vertebrate retina

The phosphoinositide (PI) cycle, discovered over 50 years ago by Mabel and Lowell Hokin, describes a series of biochemical reactions that occur on the inner leaflet of the plasma membrane of cells in response to receptor activation by extracellular stimuli. Studies from our laboratory have shown that the retina and rod outer segments (ROSs) have active PI metabolism. Biochemical studies revealed that the ROSs contain the enzymes necessary for phosphorylation of phosphoinositides. We showed that light stimulates various components of the PI cycle in the vertebrate ROS, including diacylglycerol kinase, PI synthetase, phosphatidylinositol phosphate kinase, phospholipase C, and phosphoinositide 3-kinase (PI3K). This article describes recent studies on the PI3K-generated PI lipid second messengers in the control and regulation of PI-binding proteins in the vertebrate retina.     

Phosphoinositide 3-kinase gamma controls autonomic regulation of the mouse heart through Gi-independent downregulation of cAMP level

Cardiac beta-adrenergic and the muscarinic receptors control contractility and heart rate by triggering multiple signaling events involving downstream targets like the phosphoinositide 3-kinase gamma (PI3Kgamma). We thus investigated whether the lack of PI3Kgamma could play a role in the autonomic regulation of the mouse heart. Contractility and ICaL of mutant cardiac preparations appeared increased in basal conditions and after beta-adrenergic stimulation. However, basal and beta-adrenergic stimulated heart rate were normal. Conversely, muscarinic inhibition of heart rate was reduced without alteration of the Gbetagamma-dependent stimulation of IK,ACh current. In addition, muscarinic-mediated anti-adrenergic effect on papillary muscle contractility and ICaL was significantly depressed. Consistently, cAMP level of PI3Kgamma-null ventricles was always higher than wild-type controls. Thus, PI3Kgamma controls the cardiac function by reducing cAMP concentration independently of Gi-mediated signaling.     

Somatostatin-dependent adenylyl cyclase activity in nonactivated and mitogen-activated human T cells: evidence for uncoupling of sst3 receptor from adenylyl cyclase

Neuropeptide somatostatin (SRIF) has been shown to modulate interleukin-2 (IL-2) secretion by mitogen-activated T cells. In this study, we further analyzed the transduction pathways underlying SRIF actions on human Jurkat T cells and compared SRIF signaling between nonactivated and mitogen-activated cells. SRIF effects on adenylyl cyclase activity in the absence and presence of mitogens were addressed by using three different analogs: SRIF14, SRIF28, and SMS 201-995. In semipurified membrane preparations obtained from nonactivated cells, all analogs inhibited adenylyl cyclase. However, in membrane preparations obtained from mitogen-activated cells, the maximal inhibition of adenylyl cyclase mediated by SRIF14 and SRIF28 equaled only one third of that measured in the absence of mitogens, whereas SMS 201-995 was completely inactive. To assess the relevant mechanisms associated with different effects of SRIF on adenylyl cyclase activity in nonactivated and mitogen-activated T cells, we performed binding assays by using iodinated SRIF as a radioligand. These experiments suggested that both the number of receptors and their affinities were almost identical in either nonactivated or activated cells. RT-PCR analysis of the pattern of SRIF receptor expression showed that nonactivated as well as activated Jurkat cells expressed only mRNA corresponding to the sst3 receptor subtype. Altogether, these data point to a functional activation-associated uncoupling of sst3 receptors from adenylyl cyclase in human T cells, indicating a T-cell activation-induced alteration in the sst3 receptor transduction pathway.     

Amitriptyline reduces olfactory neuron adenylyl cyclase activity

Adenylyl cyclase plays an important role in olfactory signaltransduction. Recently, a novel type III adenylyl cyclase hasbeen localized in olfactory neurons (Pfeuffer et al., 1989;Bakalyar and Reed, 1990). Because amitriptyline (AMI), a tricyclicantidepressant, appears to have an inhibitory effect on adenylylcyclase activity in other in other neuronal tissue (Yamaokaet al., 1988; Wong et al., 1991), we measured the effect ofAMI on forskolin-stimulated adenylyl cyclase activity in membranepreparations of olfactory mucosa from adult rats. In the presenceof 5'-guanylyl-imidodiphosphate, AMI (0.5–8.0 µM)inhibited forskolin-stimulated adenylyl cyclase activity ina dose-dependent manner. To determine whether this effect wasspecific for olfactory neurons, as opposed to other cells inthe olfactory epithelium, rats were unilaterally bulbectomizedin order to reduce selectively the number of olfactory neuronson the side ipsilateral to the bulbectomy. In membrane preparationsfrom unilaterally bulbectomized animals we saw significantlylower adenylyl cyclase activity in ipsilateral olfactory mucosa,compared with adenylyl cyclase activity from non-bulbectomizedmucosa. These results indicate that AMI inhibition of adenylylcyclase activity is primariy localized in olfactory neurons.     

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.     

Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption

Aldosterone, a steroid hormone, regulates renalNa⁺ reabsorption and, therefore,plays an important role in the maintenance of salt and water balance.In a model renal epithelial cell line (A6) we have found thatphosphoinositide 3-kinase (PI 3-kinase) activity is required foraldosterone-stimulated Na⁺reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na⁺ transport. Further, one of theproducts of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, isincreased in response to aldosterone in intact A6 monolayers. Thisincrease occurs just before the manifestation of the functional effectof the hormone and is also inhibited by LY-294002. With the use ofblocker-induced noise analysis, it has been demonstrated thatinhibition of phosphoinositide formation causes an inhibition ofNa⁺ entry in both control andaldosterone-pretreated cultures by reducing the number of openfunctional epithelial Na⁺ channels(ENaCs) in the apical membrane of the A6 cells. These novelobservations indicate that phosphoinositides are required for ENaCexpression and suggest a mechanism for aldosterone regulation ofchannel function.

     

Phosphoinositide 3-kinase-dependent activation of Rac

The monomeric GTPase Rac and the lipid kinase phosphoinositide 3-kinase (PI3K) are intracellular signalling enzymes that each regulate a huge range of cellular functions. Their signalling pathways overlap. Several pathways lead from PI3K activation via the production of the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P(3)) to the activation of guanine-nucleotide exchange factors (GEFs) that activate Rac. Vice versa, Rac can also stimulate the activation of PI3K, although the mechanism for this is unclear. We review here the evidence that links PI3K and Rac signalling pathways.     

Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes

The phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 decreased steady-state contraction in neonatal rat ventricular myocytes (NRVM). To determine whether the effect on steady-state contraction could be due to decreased intracellular Ca(2+) content, Ca(2+) content was assessed with fluorescent plate reader analysis by using the caffeine-releasable Ca(2+) stores as an index of sarcoplasmic reticulum (SR) Ca(2+) content. Caffeine-releasable Ca(2+) content was diminished in a dose-dependent manner with LY-294002, suggesting that the decrease in steady-state contraction was due to diminished intracellular Ca(2+) content. Activation of the L-type Ca(2+) channel by BAY K 8644 was attenuated by LY-294002, suggesting the effect of LY-294002 is to reduce Ca(2+) influx at this channel. To investigate whether additional proteins involved in excitation-contraction (EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), the ryanodine receptor, and the Na/Ca exchanger (NCX) were compared with LY-294002. Inhibition of SERCA2a by thapsigargin increased basal Ca(2+) levels in contrast to LY-294002, indicating that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine decreased SR Ca(2+) content. The additive effect with coadministration of LY-294002 could be attributed to a decrease in Ca(2+) influx at the L-type Ca(2+) channel. The NCX inhibitor Ni(2+) was used to investigate whether the decrease in intracellular Ca(2+) content with LY-294002 could be due to inhibition of the NCX reverse-mode activity. The minimal effect of LY-294002 with Ni(2+) suggests that the primary effect of LY-294002 on EC coupling occurs through inhibition of PI3K-mediated L-type Ca(2+) channel activity.     

Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis

Class I_A phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP₃). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP₃ generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.     

Phosphoinositide 3-kinase activation in late G1 is required for c-Myc stabilization and S phase entry

Phosphoinositide 3-kinase (PI3K) is one of the early-signaling molecules induced by growth factor (GF) receptor stimulation that are necessary for cell growth and cell cycle entry. PI3K activation occurs at two distinct time points during G(1) phase. The first peak is observed immediately following GF addition and the second in late G(1), before S phase entry. This second activity peak is essential for transition from G(1) to S phase; nonetheless, the mechanism by which this peak is induced and regulates S phase entry is poorly understood. Here, we show that activation of Ras and Tyr kinases is required for late-G(1) PI3K activation. Inhibition of late-G(1) PI3K activity results in low c-Myc and cyclin A expression, impaired Cdk2 activity, and reduced loading of MCM2 (minichromosome maintenance protein) onto chromatin. The primary consequence of inhibiting late-G(1) PI3K was c-Myc destabilization, as conditional activation of c-Myc in advanced G(1) as well as expression of a stable c-Myc mutant rescued all of these defects, restoring S phase entry. These results show that Tyr kinases and Ras cooperate to induce the second PI3K activity peak in G(1), which mediates initiation of DNA synthesis by inducing c-Myc stabilization.