Signalling through phosphoinositide 3-kinases: the lipids take centre stage

Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids at the 3' position of the inositol ring to generate the 3-phosphoinositides PI(3)P, PI(3,4) P2 and PI(3,4,5) P3. Recent research has shown that one way in which these lipids function in signal transduction and membrane trafficking is by interacting with 3-phosphoinositide-binding modules in a broad variety of proteins. Specifically, certain FYVE domains bind PI(3)P whereas certain pleckstrin homology domains bind PI(3,4) P2 and/or PI(3,4,5) P3. Also in 1998, PTEN - a major tumour suppressor in human cancer - was also shown to antagonise PI3K signalling by removing the 3-phosphate from 3-phosphoinositides.     

Targeting the gatekeeper residue in phosphoinositide 3-kinases

A single residue in the ATP binding pocket of protein kinases-termed the gatekeeper-has been shown to control sensitivity to a wide range of small molecule inhibitors (Chem. Biol.2004, 11, 691; Chem. Biol.1999, 6, 671). Kinases that possess a small side chain at this position (Thr, Ala, or Gly) are readily targeted by structurally diverse classes of inhibitors, whereas kinases that possess a larger residue at this position are broadly resistant. Recently, lipid kinases of the phosphoinositide 3-kinase (PI3-K) family have become the focus of intense research interest as potential drug targets (Chem. Biol.2003, 10, 207; Curr. Opin. Pharmacol.2003, 3, 426). In this study, we identify the residue that corresponds structurally to the gatekeeper in PI3-Ks, and explore its importance in controlling enzyme activity and small molecule sensitivity. Isoleucine 848 of p110alpha was mutated to alanine and glycine, but the mutated kinase was found to have severely impaired enzymatic activity. A structural bioinformatic comparison of this kinase with its yeast orthologs identified second site mutations that rescued the enzymatic activity of the I848A kinase. To probe the dimensions of the gatekeeper pocket, a focused panel of analogs of the PI3-K inhibitor LY294002 was synthesized and its activity against gatekeeper mutated and wild-type p110alpha was assessed.     

Regulation and cellular functions of class II phosphoinositide 3-kinases

Class II isoforms of PI3K (phosphoinositide 3-kinase) are still the least investigated and characterized of all PI3Ks. In the last few years, an increased interest in these enzymes has improved our understanding of their cellular functions. However, several questions still remain unanswered on their mechanisms of activation, their specific downstream effectors and their contribution to physiological processes and pathological conditions. Emerging evidence suggests that distinct PI3Ks activate different signalling pathways, indicating that their functional roles are probably not redundant. In the present review, we discuss the recent advances in our understanding of mammalian class II PI3Ks and the evidence suggesting their involvement in human diseases.     

Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes

The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.     

Gene-targeting reveals physiological roles and complex regulation of the phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3Ks) are represented by a family of eight distinct enzymes that can be divided into three classes based on their structure and function. The class I PI3Ks are heterodimeric enzymes that are regulated by recruitment to plasma membrane following receptor activation and which control numerous cellular functions, including growth, differentiation, migration, survival, and metabolism. New light has been shed on the biological role of individual members of the class I PI3Ks and their regulatory subunits through gene-targeting experiments. In addition, these experiments have brought the complexity of how PI3K activation is regulated into focus.     

Distinct phosphoinositide 3-kinases mediate mast cell degranulation in response to G-protein-coupled versus FcepsilonRI receptors

Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110beta and p85/p110delta but not p85/p110alpha are required for antigen-mediated calcium flux in RBL-2H3 cells (Smith, A. J., Surviladze, Z., Gaudet, E. A., Backer, J. M., Mitchell, C. A., and Wilson, B. S. et al., (2001) J. Biol. Chem. 276, 17213-17220). We now examine the role of Class IA PI 3-kinases isoforms in degranulation itself, using a single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110delta and p110beta blocked FcepsilonR1-mediated degranulation in response to FcepsilonRI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110alpha, despite the fact that these antibodies have no effect on antigen-induced calcium flux. These data suggest that p110alpha mediates a calcium-independent signal during degranulation. In contrast, only p110beta was required for enhancement of antigen-stimulated degranulation by adenosine, which augments mast cell-mediated airway inflammation in asthma. Finally, we examined carbachol-stimulated degranulation in RBL2H3 cells stably expressing the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly, carbachol-stimulated degranulation was blocked by antibody-mediated inhibition of the Class III PI 3-kinase hVPS34 or by titration of its product with FYVE domains. Antibodies against Class IA PI 3-kinases had no effect. These data demonstrate: (a) a calcium-independent role for p110alpha in antigen-stimulated degranulation; (b) a requirement for p110beta in adenosine receptor signaling; and (c) a requirement for hVPS34 during M1 muscarinic receptor signaling. Elucidation of the intersections between these distinct pathways will lead to new insights into mast cell degranulation.     

Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3Ks) are implicated in a variety of fundamental cellular processes. These enzymes catalyse phosphorylation of the 3'-OH position of myo-inositol lipids that serve as secondary messengers. The catalytic subunit for one of the family members, PI3K gamma, has been structurally characterized, independently, in complexes with kinase inhibitors and with the p21(Ras) GTPase. These atomic structures provide a basis for the rationalization of some PI3K substrate specificities and regulatory mechanisms, establishing links to functional and cellular data. Ongoing comprehensive structural and functional studies are essential to realize the promise of PI3K isozyme-specific therapeutic agents.     

Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.     

雷公藤对哮喘大鼠气道重构及磷脂酰肌醇3激酶表达的影响

目的:探讨雷公藤内酯醇对哮喘气道重构及磷脂酰肌醇3激酶(PI3K)表达的影响。方法:将40只SD大鼠随机分为5组(n=8):A组(正常对照组);B组(哮喘4周组);C组(哮喘6周组);D组(给药4周组);E组(给药6周组)。测定气道反应性并观察气道壁嗜酸性粒细胞浸润;图像分析软件测定支气管壁厚度、支气管平滑肌厚度及支气管平滑肌细胞核数量;免疫组织化学染色、逆转录聚合酶链式反应(RT-PCR)检测PI3K蛋白及mRNA表达。结果:①B组、C组PI3Kp85α的蛋白及mRNA表达水平显著高于A组(P均<0.01),E组上述指标较B组、C组、D组均显著降低(P<0.01、P<0.01、P<0.05);②B组及C组支气管壁厚度、支气管壁平滑肌厚度、支气管壁平滑肌细胞核数量均较A组明显增加(P均<0.01),而E组上述指标较B组、C组、D组均显著降低(P均<0.01);③B组、C组的气道反应性均高于A组(P均<0.01),E组较B组、C组、D组均显著降低(P<0.01、P<0.01、P<0.05)。结论:气道平滑肌增生是气道重构的一个显著特征,PI3K可能在此起促进作用。雷公藤内酯醇可能通过下调PI3K的表达而减轻哮喘气道高反应性及抑制气道平滑肌增生,对哮喘气道重构有一定治疗作用。     

A radioligand-binding assay for adenosine in tissue extracts

A radioligand-binding assay employing adenine analog-binding protein from rabbit crythrocytes and [³H]adenosine of high specific activity measures less than 1 pmol of adenosine in neutral HClO₄ extracts of tissue. Adenine and its nucleotides interfere with adenosine binding, so are removed by preliminary chromatography on PEI and phenyldihydroboryl cellulose columns. Free and bound adenosine are separated by filtration through cellulose acetate membranes or by absorbing free adenosine on charcoal. The recovery of 1.0–4.0 pmol of adenosine added to blood extracts and carried through purification and radioligand assay averaged 100% with an absolute error of ±0.23 pmol.     

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC–MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.     

Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence

Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.     

Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of "comet tails" that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P(2) and PI(3,4,5)P(3) on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P(2) and PI(3,4,5)P(3) and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P(2) and PI(3,4,5)P(3) formation and signaling in endomembranes.     

Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef.

A DNA probe endolabeled with digoxigenin by PCR was developed to detect and enumerate enterotoxigenic Clostridium perfringens in raw beef. After 2 h of hybridization, membranes were developed by using an anti-digoxigenin-alkaline phosphatase conjugated antibody. The resulting chromogenic reaction allowed us to detect and enumerate < or = 10 CFU of C. perfringens per g.     

SYK is upstream of phosphoinositide 3-kinase in B cell receptor signaling.

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P(3) can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P(3) production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4, 5-P(3) production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K.     

Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G(2)/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G(2)/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G(2)/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G(2)/M but have different regulatory mechanisms and function at distinct times.