The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes

The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of PtdIns(3,4,5)P3. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit PtdIns(3,4,5)P3 accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.     

Involvement of phosphoinositide 3-kinase gamma, Rac, and PAK signaling in chemokine-induced macrophage migration

In macrophages, chemotactic stimuli cause the activation of Rac and PAK, but little is known about the signaling pathways involved and their role in chemotactic gradient sensing. Herein, we report that in macrophages, the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 activates the small GTPase Rac and its downstream target PAK2 within seconds. This response depends on Gi activation and largely on the subsequent triggering of phosphoinositide 3-kinase gamma (PI3Kgamma) and Rac. Retroviral transduction of tagged Rac1 and -2 indicates that RANTES/CCL5-mediated activation of PI3Kgamma triggers Rac1 but not Rac2. In agreement, silencing of Rac1 by shRNA blocks PAK2 activity and inhibits RANTES/CCL5-induced macrophage polarization and directional migration. On the other hand, the tyrosine kinase receptor agonist CSF-1 activates PAK2 independently of PI3Kgamma and Rac. Our results thus demonstrate a chemokine-specific signaling pathway in which Gi and PI3Kgamma coordinate to drive Rac1 and PAK2 activation that eventually controls the chemotactic response.     

Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.     

Phosphoinositide 3-kinases gamma and delta, linkers of coordinate C5a receptor-Fcgamma receptor activation and immune complex-induced inflammation

Fcgamma receptors (FcgammaR) and the C5a receptor (C5aR) are key effectors of the acute inflammatory response to IgG immune complexes (IC). Their coordinated activation is critical in IC-induced diseases, although the significance of combined signaling by these two different receptor classes in tissue injury is unclear. Here we used the mouse model of the passive reverse lung Arthus reaction to define their requirements for distinct phosphoinositide 3-kinase (PI3K) activities in vivo. We show that genetic deletion of class IB PI3Kgamma abrogates C5aR signaling that is crucial for FcgammaR-mediated activation of lung macrophages. Thus, in PI3Kgamma(-/-) mice, IgG IC-induced FcgammaR regulation, cytokine release, and neutrophil recruitment were blunted. Notably, however, C5a production occurred normally in PI3Kgamma(-/-) mice but was impaired in PI3Kdelta(-/-) mice. Consequently, class IA PI3Kdelta deficiency caused resistance to acute IC lung injury. These results demonstrate that PI3Kgamma and PI3Kdelta coordinate the inflammatory effects of C5aR and FcgammaR and define PI3Kdelta as a novel and essential element of FcgammaR signaling in the generation of C5a in IC disease.     

Class IB-phosphatidylinositol 3-kinase (PI3K) deficiency ameliorates IA-PI3K-induced systemic lupus but not T cell invasion

Class I PI3K catalyzes formation of 3-poly-phosphoinositides. The family is divided into IA isoforms, activated by Tyr kinases and the IB isoform (PI3Kgamma), activated by G protein-coupled receptors. Mutations that affect PI3K are implicated in chronic inflammation, although the differential contribution of each isoform to pathology has not been elucidated. Enhanced activation of class IA-PI3K in T cells extends CD4+ memory cell survival, triggering an invasive lymphoproliferative disorder and systemic lupus. As both IA- and IB-PI3K isoforms regulate T cell activation, and activated pathogenic CD4+ memory cells are involved in triggering systemic lupus, we examined whether deletion of IB could reduce the pathological consequences of increased IA-PI3K activity. IB-PI3Kgamma deficiency did not abolish invasion or lymphoproliferation, but reduced CD4+ memory cell survival, autoantibody production, glomerulonephritis, and systemic lupus. Deletion of the IB-PI3Kgamma isoform thus decreased survival of pathogenic CD4+ memory cells, selectively inhibiting systemic lupus development. These results validate the PI3Kgamma isoform as a target for systemic lupus erythematosus treatment.     

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.     

Activation of host cell phosphatidylinositol 3-kinases by Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas' disease in humans, is an intracellular protozoan parasite with the ability to invade a wide variety of mammalian cells by a unique and remarkable process in cell biology that is poorly understood. Here we present evidence suggesting a role for the host phosphatidylinositol (PI) 3-kinases during T. cruzi invasion. The PI 3-kinase inhibitor wortmannin marked inhibited T. cruzi infection when macrophages were pretreated for 20 min at 37 degrees C before inoculation. Infection of macrophages with T. cruzi markedly stimulated the formation of the lipid products of the phosphatidylinositol (PI) 3-kinases, PI 3-phospate, PI 3,4-biphosphate, and PI 3,4,5-triphosphate, but not PI 4-phosphate or PI 4,5-biphosphate. This activation was inhibited by wortmannin. Infection with T. cruzi also stimulated a marked increase in the in vitro lipid kinase activities that are present in the immunoprecipitates of anti-p85 subunit of class I PI 3-kinase and anti-phosphotyrosine. In addition, T. cruzi invasion also activated lipid kinase activity found in immunoprecipitates of class II and class III PI 3-kinases. These data demonstrate that T. cruzi invasion into macrophages strongly activates separated PI 3-kinase isoforms. Furthermore, the inhibition of the class I and class III PI 3-kinase activities abolishes the parasite entry into macrophages. These findings suggest a prominent role for the host PI 3-kinase activities during the T. cruzi infection process.     

Activation of phosphoinositide 3-kinase gamma by Ras

BACKGROUND: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation.RESULTS: We show that posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3Kgamma could we detect any evidence of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate.CONCLUSIONS: Our data suggest that Ras activate PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.     

Dissociation of apoptosis from proliferation, protein kinase B activation, and BAD phosphorylation in interleukin-3-mediated phosphoinositide 3-kinase signaling

Interleukin-3 (IL-3) acts as both a growth and survival factor for many hemopoietic cells. IL-3 treatment of responsive cells leads to the rapid and transient activation of Class IA phosphoinositide-3-kinases (PI3Ks) and the serine/threonine kinase Akt/protein kinase B (PKB) and phosphorylation of BAD. Each of these molecules has been implicated in anti-apoptotic signaling in a wide range of cells. Using regulated expression of dominant-negative p85 (Deltap85) in stably transfected IL-3-dependent BaF/3 cells, we have specifically investigated the role of class IA PI3K in IL-3 signaling. The major functional consequence of Deltap85 expression in these cells is a highly reproducible, dramatic reduction in IL-3-induced proliferation. Expression of Deltap85 reduces IL-3-induced PKB phosphorylation and activation and phosphorylation of BAD dramatically, to levels seen in unstimulated cells. Despite these reductions, the levels of apoptosis observed in the same cells are very low and do not account for the reduction in IL-3-dependent proliferation we observe. These results show that Deltap85 inhibits both PKB activity and BAD phosphorylation without significantly affecting levels of apoptosis, suggesting that there are targets other than PKB and BAD that can transmit survival signals in these cells. Our data indicate that the prime target for PI3K action in IL-3 signaling is at the level of regulation of proliferation.     

Stromal cell-derived factor-1alpha induces tube-like structure formation of endothelial cells through phosphoinositide 3-kinase

Stromal cell-derived factor-1alpha (SDF-1alpha) is a CXC chemokine, which induces tube formation of endothelial cells. Although SDF-1alpha transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1alpha-mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1alpha-mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (immortalized murine brain capillary endothelial) cells). SDF-1alpha stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and G(i) protein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1alpha activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted Deltap85-8 cells), which lacks association with the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1alpha failed to activate PI3-kinase and to stimulate tube formation. SDF-1alpha-induced tube formation was inhibited by an antibody against murine vascular endothelial cadherin. The antibody as well as LY294002 attenuated SDF-1alpha-mediated compact cell-cell contact, which proceeded to tube formation. Taken together, SDF-1alpha induces compact cell-cell contact through PI3-kinase, resulting in tube formation of endothelial cells.     

Class II phosphoinositide 3-kinase is activated by insulin but not by contraction in skeletal muscle.

While the role of the class IA phosphoinositide 3-kinase (PI 3-kinase) in insulin signaling is well established, little is known about the role of the class II PI 3-kinases. We show that insulin stimulation of intact rat soleus and epitrochlearis muscles causes a 3- to 4-fold increase in the activity of the wortmannin-resistant alpha isoform of the class II PI 3-kinase (PI3K-C2alpha). This activation is rapid and parallels the insulin-induced activation of the class IA PI 3-kinase associated with IRS-1 in these muscles. However, while contraction activated p38 Map kinase, it did not stimulate the activity of the class II PI 3-kinase. Therefore, activation of class II PI 3-kinase is unlikely to provide a mechanism that explains the fact that exercise-induced activation of glucose uptake is not blocked by wortmannin. However, the results suggest that activation of class II PI 3-kinase is likely to play a role in insulin signaling pathways in skeletal muscle.     

Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1alpha: implications for leukocyte transmigration

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.     

A high-throughput liposome substrate assay with automated lipid extraction process for PI 3-kinase

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P(2) to PI (3,4,5)P(3), which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110alpha, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases.     

Direct interaction of major histocompatibility complex class II-derived peptides with class Ia phosphoinositide 3-kinase results in dose-dependent stimulatory effects

Peptides corresponding to residues 65-79 of human lymphocyte antigen class II sequence (DQA*03011) are cell-permeable and at high concentrations block activation of protein kinase B/Akt and p70-S6 kinase in T-cells, effects attributed to inhibition of phosphoinositide (PI) 3-kinase activity. To understand the molecular basis of this, we analyzed the effect this peptide had on activity of class I PI 3-kinases. Although there was no effect on the activity of class Ib PI 3-kinase or on the protein kinase activity of class I PI 3-kinases, there was a biphasic effect on lipid kinase activity of the class Ia enzymes. There was an inhibition of activity at higher peptide concentrations because of a formation of insoluble complexes between peptide and enzyme. Conversely, at lower peptide concentrations there was a profound activation of PI 3-kinase activity of class Ia PI 3-kinases. Studies of peptide variants revealed that all active peptides conform to heptad repeat motifs characteristic of coiled-coil helices. Surface plasmon resonance studies confirmed direct sequence-specific binding of active peptide to the p85alpha adapter subunit of class Ia PI 3-kinase. Active peptides also activated protein kinase B and extracellular signal-regulated kinase (ERK) in vivo in a wortmannin-sensitive manner while reducing recoverable cellular p85 levels. These results indicate that the human lymphocyte antigen class II-derived peptides regulate PI 3-kinase by direct interaction, probably via the coiled-coil domain. These peptides define a novel mechanism of regulating PI 3-kinase and will provide a useful tool for specifically dissecting the function of class Ia PI 3-kinase in cells and for probing structure-function relationships in the class Ia PI 3-kinase heterodimers.     

Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by inhibition of PI3-kinase

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.     

Phosphoinositide 3-kinases can act independently of p27Kip1 to regulate optimal IL-3-dependent cell cycle progression and proliferation

We have examined the role of phosphoinositide 3-kinases (PI3K) in interleukin (IL)-3-dependent cell cycle progression and compared the effects of LY294002 with expression of a dominant negative form of p85, termed Deltap85, which more specifically inhibits class I(A) PI3Ks. Inhibition of PI3Ks in BaF/3 led to accumulation of cells in G1 and extension of cell cycle transit times. Biochemically, both LY294002 and Deltap85 decreased levels of p107 and cyclins D2, D3 and E and reduced retinoblastoma protein (pRb) phosphorylation. Significantly, only LY294002 treatment increased expression of p27(Kip1). Interestingly, LY294002 decreased IL-3-induced proliferation of primary bone marrow-derived mast cells (BMMC) derived from both wild-type and p27(Kip1)-deficient mice and importantly, LY294002 treatment failed to upregulate p27(Kip1) in wild-type BMMC. These data support a role for class I(A) PI3K in regulating optimal cell cycle progression in response to IL-3 and demonstrate that upregulation of p27(Kip1) is not essential for attenuation of the cell cycle resulting from PI3K inhibition.     

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.     

Intracellular Signal Cascade in CD4<Superscript>+</Superscript>T-Lymphocyte Migration Stimulated by Interferon-γ-Inducible Protein-10

The intracellular signal cascades involved in chemokine-stimulated migration of in vitro activated human peripheral blood CD4+ T-lymphocytes were investigated. IP-10-mediated chemotactic response of lymphocytes was decreased in the presence of selective inhibitors of Src-kinases (by 40-45%), PI3-kinases (35-40%), and MAP-kinases ERK1/2 (35-40%) and p38 (20%). Combined addition of specific inhibitors of Src-kinases and PI3-kinases and inhibitors of Src-kinases and ERK1/2 MAP-kinases did not result in the further increase of the inhibitory effect, while the combined addition of specific inhibitors of PI3-kinases and ERK1/2 MAP-kinases decreased migration of CD4+ T-lymphocytes more effectively (by 55-60%) than any individual inhibitor. Immunoblotting analysis of activation of MAP-kinases ERK1/2 and p38 revealed increased level of phosphorylation of ERK1/2 and p38 MAP-kinases in the presence IP-10. Selective inhibitors of Src-kinases and PI3-kinases significantly inhibited phosphorylation of p38 but did not influence phosphorylation of ERK1/2 MAP-kinases. Our results suggest that Src-kinases, PI3-kinases, and ERK1/2 MAP-kinases are involved in intracellular signal cascade activated during IP-10-stimulated migration of T-lymphocytes, whereas p38 MAP-kinases do not participate in the migration process, although its activation induced by IP-10 depends on Src-kinases and PI3-kinases.     

Anionic phospholipids are involved in membrane targeting of PI 3-kinase gamma

Phosphoinositide 3-kinases (PI 3-kinases) have critical roles in diverse cellular signaling processes and in protein trafficking. In contrast to the class I PI 3-kinases alpha, beta, and delta which bind via src homology 2 (SH2) domains of adaptor proteins to tyrosine kinase receptors, the mechanism of recruitment of the PI 3-kinase gamma to membranes is unknown. We report in vitro experiments using immobilized proteins and small unilamellar vesicles which suggest an involvement of anionic phospholipids in membrane association of PI 3-kinase gamma. Furthermore we provide evidence that the enzyme displays beside the catalytic center a phospholipid binding domain which is essential for enzyme activity.