Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.     

Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.     

PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2)

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.     

Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.     

A membrane-permeant ester of phosphatidylinositol 3,4, 5-trisphosphate (PIP(3)) is an activator of human neutrophil migration

Activity of phosphatidylinositol (PI) 3-kinase is required for optimal migration of human neutrophils [Niggli and Keller (1999) Eur. J. Pharmacol. 335, 43-52]. We have tested the direct effect of a product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), on neutrophil migration. To this end, a membrane-permeant ester of PIP(3), dilauroyl phosphatidylinositol 3,4, 5-trisphosphate-heptakis-(acetooxymethyl)ester (PIP(3)/AM) was used. PIP(3)/AM (ED(50): 10-17 microM) induced development of polarity and accumulation of F-actin in the leading lamellae in up to 70% of the cells. These cells exhibited stimulated random migration, comparable to that observed in uniform concentrations of chemotactic peptide. Evidence is provided for a role of Rho-kinase and for activation of PI 3-kinase in a positive feedback loop in PIP(3)/AM-induced motility.     

Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN

The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.     

磷脂酰肌醇3-激酶γ/δ与炎症相关疾病

磷脂酰肌醇3-激酶(PI3K)是一类脂质与蛋白激酶家族,其主要通过在磷脂酰肌醇的肌醇环三位进行磷酸化产生胞内重要的第二信使——磷脂酰肌醇-3,4,5-三磷酸(phosphatidyl inositol 3,4,5-trisphosphate,PIP3)而发挥作用.磷脂酰肌醇3-激酶γ/δ(PI3Kγ/δ)是I类PI3K家族中的成员,其主要表达于免疫相关细胞中,这2种PI3K亚型参与先天性与获得性免疫应答.因此,PI3Kγ/PI3Kδ被视为因免疫反应调控异常导致的炎症疾病的治疗药物靶点.目前,利用特异性抑制剂靶向干预PI3Kγ和/或PI3Kδ,成为炎症相关疾病治疗的新策略.本文简介了PI3Kγ与PI3Kδ在不同类型免疫细胞中的功能;并就采用小分子特异性抑制剂,靶向抑制PI3Kγ和/或PI3Kδ在各类炎症相关疾病中的治疗作用和效果进行综述.     

Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates

Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.     

PIP3 is involved in neuronal polarization and axon formation

Recent experiments in various cell types such as mammalian neutrophils and Dictyostelium discoideum amoebae point to a key role for the lipid product of PI 3-kinase, PIP(3), in determining internal polarity. In neurons, as a consequence of the elongation of one neurite, the axon is specified and the cell acquires its polarity. To test the hypothesis that PI 3-kinase and PIP(3) may play a role in neuronal polarity, and especially in axon specification, we observed localization of PIP(3) visualized by Akt-PH-GFP in developing hippocampal neurons. We found that PIP(3) accumulates in the tip of the growing processes. This accumulation is inhibited by addition of PI 3-kinase inhibitors. Those inhibitors, consistently with a role of PIP(3) in process formation and elongation, delay the transition from stage 1 neurons to stage 3 neurons, and both axon formation and elongation. Moreover, when the immature neurite contacts a bead coated with laminin, a substrate known to induce axon specification, PIP(3) accumulates in its growth cone followed by a rapid elongation of the neurite. In such conditions, the addition of PI 3-kinase inhibitors inhibits both PIP(3) accumulation and future axon elongation. These results suggest that PIP(3) is involved in axon specification, possibly by stimulating neurite outgrowth. In addition, when a second neurite contacted the beads, this neurite rapidly elongates whereas the elongation of the first laminin-contacting neurite stops, consistently with the hypothesis of a negative feedback mechanism from the growing future axon to the other neurites.     

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.     

A PI3-kinase signaling code for insulin-triggered insertion of glucose transporters into the plasma membrane

Activation of phosphatidyl-inositol-3'-OH-kinase (PI3K) and the resulting production of phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) are ubiquitous signaling steps that link various cell surface receptors to multiple intracellular targets. In fat and muscle cells, the same PI3K pathway that regulates metabolic enzymes, proliferation, and differentiation has also been shown to be involved in insulin-triggered insertion of glucose transporter GLUT4 into the plasma membrane. The multiple PI3K functions raise the question of how the same PI3K pathway can be selectively used for different cell functions. Here we developed a dual-color evanescent wave microscopy method to simultaneously measure PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes. Activation of PI3K was found to be both necessary and sufficient for triggering GLUT4 insertion, but transporter insertion was markedly suppressed for small-amplitude, persistent PIP3 signals and for large-amplitude, short PIP3 signals. The rejection of these common PI3K signaling responses may explain the selective advantage of insulin over platelet-derived growth factor and other stimuli for inducing GLUT4 insertion. Our study suggests that the same PI3K pathway can control specific cell functions by relying on effector systems that respond to particular receptor-encoded time courses and amplitudes of PIP3 signals.     

Escherichia coli K-1 interaction with human brain micro-vascular endothelial cells triggers phospholipase C-gamma1 activation downstream of phosphatidylinositol 3-kinase

Escherichia coli, the most common Gram-negative bacterium that causes meningitis in neonates, invades human brain microvascular endothelial cells (HBMEC) by rearranging host cell actin via the activation of phosphatidylinositol 3-kinase (PI3K) and PKC-alpha. Here, further, we show that phospholipase (PLC)-gamma1 is phosphorylated on tyrosine 783 and condenses at the HBMEC membrane beneath the E. coli entry site. Overexpression of a dominant negative (DN) form of PLC-gamma, the PLC-z fragment, in HBMEC inhibits PLC-gamma1 activation and significantly blocks E. coli invasion. PI3K activation is not affected in PLC-z/HBMEC upon infection, whereas PKC-alpha phosphorylation is completely abolished, indicating that PLC-gamma1 is downstream of PI3K. Concomitantly, the phosphorylation of PLC-gamma1 is blocked in HBMEC overexpressing a dominant negative form of the p85 subunit of PI3K but not in HBMEC overexpressing a dominant negative form of PKC-alpha. In addition, the recruitment of PLC-gamma1 to the cell membrane in both PLC-z/HBMEC and DN-p85/HBMEC is inhibited. Activation of PI3K is associated with the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 1,4,5-trisphosphate (PIP3), which in turn recruits PLC-gamma1 to the cell membrane via its interaction with pleckstrin homology domain of PLC-gamma1. Utilizing the pleckstrin homology domains of PKC-delta and Btk proteins fused to green fluorescent protein (GFP), which specifically interact with PIP2 and PIP3, respectively, we show herein that E. coli invasion induces the breakdown of PIP2 at the plasma membrane near the site of E. coli interaction. PIP3, on the other hand, recruits the GFPBkt to the cell membrane beneath the sites of E. coli attachment. Our studies further show that E. coli invasion induces the release of Ca2+ from intracellular pools as well as the influx of Ca2+ from the extracellular medium. This elevation in Ca2+ levels is completely blocked both in PLC-z/HBMEC and DN-p85/HBMEC, but not in DN-PKC/HBMEC. Taken together, these results suggest that E. coli infection of HBMEC induces PLC-gamma1 activation in a PI3K-dependent manner to increase Ca2+ levels in HBMEC. This is the first report demonstrating the recruitment of activated PLC-gamma1 to the sites of bacterial entry.     

ACTH stimulates turnover of the phosphatidylinositol-glycan

Primary cultures of calf adrenal glomerulosa cells were prelabeled for 3 days with [3H]inositol or [3H]glucosamine and stimulated with 10 nM ACTH. Labeled phosphatidylinositol (PI), polyphosphoinositides (PIP and PIP2) and a novel phosphatidylinositol-glycan (PI-glycan) were measured after separation by TLC. [3H]-Inositol labeling of PI, PIP and PIP2 increased rapidly, whereas labeling of the PI-glycan showed an initial decrease at 1 minute followed by a subsequent increase. Similar results were obtained when cells were prelabeled with [3H]glucosamine, viz. the PI-glycan label decreased at 1 min and subsequently increased. These results suggest that ACTH provokes (a) coordinated increases in the synthesis of PI, PIP, PIP2 and the PI-glycan, and (b) the increase in PI-glycan synthesis is preceded by initial decrease, presumably reflecting hydrolysis of this lipid.     

New Functions for PI3K in the Control of Cell Division

Although cell lipids were initially envisioned as structural components of the cell, their essential contribution to initiation and regulation of cell responses is now clearly established. Among the different lipids that regulate cell responses, those produced by class I phosphoinositide 3-kinase (PI3K), phosphatidylinositol (3,4)P₂ (PIP₂), and phosphatidylinositol (3,4,5)P₃ (PIP₃), have concentrated much attention in recent years. PIP2 and PIP3 are involved in cell division and survival control, and mutations in the PI3K pathway are linked to autoimmunity and cancer. Here we discuss two novel observations: a PI3K function in the late-G₁ phase of the cell cycle and the contribution of the p85 PI3K regulatory subunit in the control of cytokinesis.     

Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration

Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) production, a polarized distribution of PIP(3) was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP(3) or induce cell polarization. Thus, the increase in PIP(3) concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.     

Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.     

The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling

Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.     

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Chemotaxis in the absence of PIP3 gradients

Chemotaxing neutrophils and Dictyostelium amoebae produce in their plasma membranes the signaling lipid PI(3,4,5)P3 (PIP3) in gradients, which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene-knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is, however, a consistent defect in movement speed in chemotaxis and especially in random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but it can affect cell speed. A signaling pathway from receptor to cytoskeleton able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases must exist.