Structural insights into phosphoinositide 3-kinase catalysis and signalling

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.     

EPS8 and E3B1 transduce signals from Ras to Rac.

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.     

Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase.

Although the hormone-stimulated synthesis of 3-phosphorylated inositol lipids is known to form an intracellular signalling system, there is no consensus on the crucial receptor-regulated event in this pathway and it is still not clear which of the intermediates represent potential output signals. We show here that the key step in the synthesis of 3-phosphorylated inositol lipids in 3T3 cells stimulated by platelet-derived growth factor is the activation of a phosphatidylinositol(4,5)-bisphosphate (3)-hydroxy (PtdIns(4,5)P2 3-OH) kinase. A similar conclusion has been applied to explain the actions of formyl-Met-Leu-Phe on neutrophils, and it may be that receptors that couple through intrinsic tyrosine kinases or through G proteins stimulate the same step in 3-phosphorylated inositol lipid metabolism. The close parallel between these two mechanisms for the activation of PtdIns(4,5)P2 3-OH kinase and those described for the activation of another key signalling enzyme, phospholipase C (ref. 7), focuses attention on the product of the PtdIns(4,5)P2 3-OH kinase, PtdIns(3,4,5)P3, as a possible new second messenger.     

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.     

运动细胞初始极化阶段胞内信号分子双向积聚的分子机制及动态数值模拟

真核细胞（Eukaryotes）,如盘基网柄菌细胞（Dictyo stelium）和白细胞（Leukocyte）等,受到前方传来的外信号刺激后,胞内物质发生两种方向相反的运动：胞质中与质膜结合的磷酸激酶PI3K及其在质膜上的生成物磷脂酰肌醇-3,4,5-三磷酸PI（3,4,5）P3以向前扩散的方式积聚到前沿;而胞质中与质膜结合的磷酸酶PTEN及其在质膜上的生成物磷脂酰肌醇-4,5-双磷酸PI（4,5）P2以向后扩散的方式积聚到质膜的后边.通过这种＂双向积聚＂,细胞形成头部和尾部,并在前沿生出伪足,完成初始极化.本文根据已知的实验,分析了＂双向积聚＂的分子机制,建立了相应的数学模型,通过数值模拟加以分析.以胞内被激活的小G蛋白（Rac）为触发信号,其梯度与外信号场的梯度一致;PI3K和PTEN作为调控因子,PI（3,4,5）P3和PI（4,5）P2作为标识细胞极化的信号分子,它们的浓度变化由一组耦合的非稳态二维反应-扩散方程描述.该反应-扩散体系源项中包含：PI（3,4,5）P3对PI3K,PI（4,5）P2对PTEN的识别和结合过程,是由蒙特-卡诺（Monte-Carlo）法处理;质膜结合态PI3K和PTEN对PI（3,4,5）P3和PI（4,5）P2施加的酶催化作用,由Michaelis-Menten动力学过程描述.反应-扩散方程组采用格子Boltzmann方法进行数值求解.数值试验显示,产生＂双向积聚＂的关键是受外信号梯度刺激后的胞内信号分子相互激发或抑制所形成的正反馈或负反馈回路：给细胞质膜头部一个较高的Rac激活率,Rac→PI3K？PI（3,4,5）P3将形成短程正反馈回路（亦即＂局部激励＂）,引起PI3K和PI（3,4,5）P3快速在细胞头部积聚;头部PI（3,4,5）P3增多,限制了PTEN与PI（4,5）P2结合,使得PI（3,4,5）P3？PTEN→PI（4,5）P2形成长程负反馈回路（亦即＂全局抑制＂）;引起PTEN和PI（4,5）P2慢慢在细胞尾部积聚.同时发现,PI3K和PTEN含量对细胞极化有明显的影响,并存在使细胞正确极化的最佳值.     

Ras, PI(3)K and mTOR signalling controls tumour cell growth

All eukaryotic cells coordinate cell growth with the availability of nutrients in their environment. The mTOR protein kinase has emerged as a critical growth-control node, receiving stimulatory signals from Ras and phosphatidylinositol-3-OH kinase (PI(3)K) downstream from growth factors, as well as nutrient inputs in the form of amino-acid, glucose and oxygen availability. Notably, components of the Ras and PI(3)K signalling pathways are mutated in most human cancers. The preponderance of mutations in these interconnected pathways suggests that the loss of growth-control checkpoints and promotion of cell survival in nutrient-limited conditions may be an obligate event in tumorigenesis.     

Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3

Oncogenic tyrosine kinases have proved to be promising targets for the development of highly effective anticancer drugs. However, tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (HER) family show only limited activity against HER2-driven breast cancers, despite effective inhibition of epidermal growth factor receptor (EGFR) and HER2 in vivo. The reasons for this are unclear. Signalling in trans is a key feature of this multimember family and the critically important phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 (refs 9, 10). Here we show that HER3 and consequently PI(3)K/Akt signalling evade inhibition by current HER-family TKIs in vitro and in tumours in vivo. This is due to a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium, driven by increased membrane HER3 expression driving the phosphorylation reaction and by reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt-mediated negative-feedback signalling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abrogation of HER3 resistance by small interfering RNA knockdown restores potent pro-apoptotic activity to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumours and the therapeutic promise of this oncoprotein target. However, because HER3 signalling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to silence oncogenic HER2 signalling effectively. The biologic marker with which to assess the efficacy of HER TKIs should be the transphosphorylation of HER3 rather than autophosphorylation.     

Spred is a Sprouty-related suppressor of Ras signalling

Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.     

Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane.     

Cbl-b regulates the CD28 dependence of T-cell activation

Whereas co-stimulation of the T-cell antigen receptor (TCR) and CD28 triggers T-cell activation, stimulation of the TCR alone may result in an anergic state or T-cell deletion, both possible mechanisms of tolerance induction. Here we show that T cells that are deficient in the adaptor molecule Cbl-b (ref. 3) do not require CD28 engagement for interleukin-2 production, and that the Cbl-b-null mutation (Cbl-b(-/-)) fully restores T-cell-dependent antibody responses in CD28-/- mice. The main TCR signalling pathways, such as tyrosine kinases Zap-70 and Lck, Ras/mitogen-activated kinases, phospholipase Cgamma-1 and Ca2+ mobilization, were not affected in Cbl-b(-/-) T cells. In contrast, the activation of Vav, a guanine nucleotide exchange factor for Rac1/Rho/CDC42, was significantly enhanced. Our findings indicate that Cbl-b may influence the CD28 dependence of T-cell activation by selectively suppressing TCR-mediated Vav activation. Mice deficient in Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis, suggesting that the dysregulation of signalling pathways modulated by Cbl-b may also contribute to human autoimmune diseases such as multiple sclerosis.     

Two polyphosphatidylinositide metabolites control two K+ currents in a neuronal cell

Hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) produces two prospective intracellular messengers: inositol 1,4,5-trisphosphate (InsP3), which releases Ca2+ from intracellular stores; and diacylglycerol (DG), which activates protein kinase C. Here we show how the formation of these two substances triggered by one external messenger, bradykinin, leads to the appearance of two different sequential membrane conductance changes in the neurone-like NG108-15 neuroblastoma-glioma hybrid cell line. In these cells bradykinin rapidly hydrolyses PtdIns(4,5)P2 to InsP3 and DG, raises intracellular Ca2+ and hyperpolarizes then depolarizes the cell membrane. By voltage-clamp recording we show that the hyperpolarization results from the activation pharmacologically-identifiable species of Ca2+-dependent K+ current. This is also activated by intracellular injections of Ca2+ or InsP3 so may be attributed to the formation and action of InsP3. The subsequent depolarization results primarily from the inhibition of a different, voltage-dependent K+ current, the M-current that is also inhibited by DG activators. Hence we describe for the first time a dual, time-dependent role for these two intracellular messengers in the control of neuronal signalling by a peptide.     

Phospholipase C gamma 1 is a physiological guanine nucleotide exchange factor for the nuclear GTPase PIKE

Phospholipase C gamma 1 (PLC-gamma 1) hydrolyses phosphatidylinositol-4,5-bisphosphate to the second messengers inositol-1,4,5-trisphosphate and diacylglycerol. PLC-gamma 1 also has mitogenic activity upon growth-factor-dependent tyrosine phosphorylation; however, this activity is not dependent on the phospholipase activity of PLC-gamma 1, but requires an SH3 domain. Here, we demonstrate that PLC-gamma 1 acts as a guanine nucleotide exchange factor (GEF) for PIKE (phosphatidylinositol-3-OH kinase (PI(3)K) enhancer). PIKE is a nuclear GTPase that activates nuclear PI(3)K activity, and mediates the physiological activation by nerve growth factor (NGF) of nuclear PI(3)K activity. This enzymatic activity accounts for the mitogenic properties of PLC-gamma 1.     

Oncogenic kinase signalling

Protein-tyrosine kinases (PTKs) are important regulators of intracellular signal-transduction pathways mediating development and multicellular communication in metazoans. Their activity is normally tightly controlled and regulated. Perturbation of PTK signalling by mutations and other genetic alterations results in deregulated kinase activity and malignant transformation. The lipid kinase phosphoinositide 3-OH kinase (PI(3)K) and some of its downstream targets, such as the protein-serine/threonine kinases Akt and p70 S6 kinase (p70S6K), are crucial effectors in oncogenic PTK signalling. This review emphasizes how oncogenic conversion of protein kinases results from perturbation of the normal autoinhibitory constraints on kinase activity and provides an update on our knowledge about the role of deregulated PI(3)K/Akt and mammalian target of rapamycin/p70S6K signalling in human malignancies.     

Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum

Phosphatidylinositol (PtdIns), the most abundant phosphoinositide, is the precursor of phosphatidylinositol 4-monophosphate which is converted to phosphatidylinositol 4,5-bisphosphate, the lipid hydrolysed as an early step in signal transduction by many stimuli. It is generally thought that a single enzyme in the endoplasmic reticulum, PtdIns synthase (CDP-diglyceride:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11), is responsible for PtdIns synthesis and that newly synthesized PtdIns is transported to the plasma membrane by exchange proteins. Several investigators have proposed that there are two functionally distinct pools of PtdIns, one responsive to stimulation and the other not, and that the stimulus-responsive pool may be synthesized at a different site within the cell, perhaps within the plasma membrane. Indeed, it was suggested that there is PtdIns synthase activity in plasma membrane isolated from rat liver. GH3 rat pituitary tumour cells are an excellent model system to study stimulation of phosphoinositide metabolism by thyrotropin-releasing hormone (TRH). Conversion of PtdIns to polyphosphoinositides and TRH (and GTP)-activated phosphoinositide hydrolysis are known to occur in plasma membrane isolated from GH3 cells. Here we report that PtdIns synthase activity in the plasma membrane of GH3 cells is distinct from that present in the endoplasmic reticulum. The plasma membrane PtdIns synthase may be responsible for a portion of PtdIns re-synthesis that occurs during cell stimulation.     

The inositol tris/tetrakisphosphate pathway--demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues

Recent advances in our understanding of the role of inositides in cell signalling have led to the central hypothesis that a receptor-stimulated phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) results in the formation of two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The existence of another pathway of inositide metabolism was first suggested by the discovery that a novel inositol trisphosphate, Ins(1,3,4)P3, is formed in stimulated tissues; the metabolic kinetics of Ins(1,3,4)P3 are entirely different from those of Ins(1,4,5)P3 (refs 6, 7). The probable route of formation of Ins(1,3,4)P3 was recently shown to be via a 5-dephosphorylation of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a compound which is rapidly formed on muscarinic stimulation of brain slices, and which can be readily converted to Ins(1,3,4)P3 by a 5-phosphatase in red blood cell membranes. However, the source of Ins(1,3,4,5)P4 is unclear, and an attempt to detect a possible parent lipid, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), was unsuccessful. The recent discovery that the higher phosphorylated forms of inositol (InsP5 and InsP6) also exist in animal cells suggested that inositol phosphate kinases might not be confined to plant and avian tissues, and here we show that a variety of animal tissues contain an active and specific Ins(1,4,5)P3 3-kinase. We therefore suggest that an inositol tris/tetrakisphosphate pathway exists as an alternative route to the dephosphorylation of Ins(1,4,5)P3. The function of this novel pathway is unknown.     

Essential role for Gab2 in the allergic response.

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.     

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans

The coordination of signals from different pathways is important for cell fate specification during animal development. Here, we define a novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p (ref. 2), the VPC believed to be the source of the lateral signal. Here we identify a 'downregulation targeting signal' (DTS) in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. We also show that if LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo.