Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β₂ integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β₂ integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr³²³) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I_A. Syk-Tyr³²³ was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class I_A as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β₂ integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β₂ integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Implication of phosphatidylinositol 3-kinase membrane recruitment in hydrogen peroxide-induced activation of PI3K and Akt

The effect of tyrosine phosphorylation of PI3K on its enzymatic activity is quite controversial, and the molecular mechanism by which ROS trigger PI3K membrane relocation is unclear. Therefore, we investigated the regulatory mechanism of hydrogen peroxide-induced PI3K activation in DT40 cells, utilizing genetic and pharmacological approaches. Our results revealed that hydrogen peroxide induced tyrosine phosphorylation of the p110 but not the p85 subunit of PI3K in DT40 cells. This phosphorylation was intact in Btk- and Cbl-deficient DT40 cells, but was drastically suppressed in Lyn, Syk, or BCAP-deficient DT40 cells. Tyrosine phosphorylation of p110 did not alter its catalytic activity, and hydrogen peroxide stimulation did not cause an increase in the intrinsic PI3K activity; however, hydrogen peroxide stimulation did induce PI(3,4,5)P3 accumulation and activate Akt. The activation of Akt, as monitored by its ability to phosphorylate GSK-3alpha/beta and by its S473 phosphorylation, was strictly dependent on PI3K activity. Under our conditions, hydrogen peroxide-induced PI3K and Akt activation was independent of Lyn, Syk, Cbl, BCAP, or Ras when each was eliminated individually either by mutation or by a specific inhibitor. In comparison, Akt activation by B cell receptor cross-linking was dependent on BCAP. In addition, hydrogen peroxide treatment caused an increase in the amount of p85 PI3K associated with the particulate fraction. Together, these results indicate that the hydrogen peroxide-induced PI3K and Akt activation in DT40 cells was achieved through PI3K membrane recruitment to its substrate site, thereby enabling PI3K to maximize its catalytic efficiency.     

The non-receptor tyrosine kinase Syk regulates lamellipodium formation and site-directed migration of human leukocytes

The tyrosine kinase Syk is associated with CD18, the beta-subunit of the leukocyte adhesion molecules of the beta(2) integrin family (CD11/CD18), and becomes activated upon beta(2) integrin-mediated adhesion. In this study, we elucidated the role of Syk in polarization and site-directed migration of neutrophil-like differentiated HL-60 cells and monocytic THP-1 cells. By means of confocal microscopy, we detected a homogenous distribution of Syk in unstimulated cells in suspension. The stimulation of HL-60 cells by formyl-methionyl-leucyl-phenylalanine (fMLP, 100 nM) or the activation of THP-1 cells by monocyte chemoattractant protein-1 (10 ng/ml) induced beta(2) integrin-mediated cell adhesion and polarization on immobilized fibrinogen which was associated with an enrichment of Syk at the lamellipodium forming site. This effect was abolished by function blocking anti-CD18 antibody or by treatment of the cells with the Syk inhibitor piceatannol (30 microM) suggesting that the redistribution of Syk required both, beta(2) integrin-mediated adhesion and Syk activation. Moreover, the inhibition of Syk by piceatannol or the downregulation of Syk by antisense technique resulted in an excessive formation of lamellipodia indicating that Syk may act as a negative regulator that limits lamellipodium formation. The analysis of chemotaxis revealed that the inhibition of Syk impaired the ability of the cells to follow a chemotactic gradient whereas random migration was intact. Taken together, our data suggest a novel role for Syk in the maintenance of a bipolar phenotype by regulating lamellipodium formation, which is a critical prerequisite for site-directed migration of leukocytes.     

Class I phosphoinositide 3-kinase p110beta is required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by macrophages

Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110alpha, p110beta, and p110delta and one gene for the class IB p110 subunit encoding p110gamma. Here we report a specific recruitment of p110beta and p110delta (but not p110alpha) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110beta is the major isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110delta showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110delta in this process. Similarly, p110gamma-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110beta is the major class I catalytic isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary macrophages.     

Membrane localization of all class I PI 3-kinase isoforms suppresses c-Myc-induced apoptosis in Rat1 fibroblasts via Akt

Phosphoinositide 3'-kinases (PI3Ks) constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. PI3Ks are heterodimers made up of four different 110-kDa catalytic subunits (p110alpha, p110beta, p110gamma, and p110delta) and a smaller regulatory subunit. Despite a clear implication of PI3Ks in survival signaling, the contribution of the individual PI3K isoforms has not been elucidated. To address this issue, we generated Rat1 fibroblasts that co-express c-Myc and membrane targeted derivates of the different p110 isoforms. Here we present data for the first time showing that activation of PI3-kinase signaling through membrane localization of p110beta, p110gamma, and p110delta protects c-Myc overexpressing Rat1 fibroblasts from apoptosis caused by serum deprivation like it has been described for p110alpha. Expression of each p110 isoform reduces significantly caspase-3 like activity in this apoptosis model. Decreased caspase-3 activity correlates with the increase in Akt phosphorylation in cells that contain one of the myristoylated p110 isoforms. p110 isoform-mediated protection from cell death was abrogated upon expression of a kinase-negative version of Akt.     

A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils

During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space.     

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.     

Human platelets contain p110delta phosphoinositide 3-kinase

The phosphoinositide 3-kinase (PI3K) catalytic subunit p110delta, the most recently discovered member of the heterodimeric Class IA PI3K family, has been detected uniquely in leukocytes, but not in one member of the leukocyte family: platelets. We have examined freshly prepared isolates of human platelets for the presence of this enzyme, realizing that p110delta is highly susceptible to proteolytic degradation. We have utilized p110delta-directed Western blotting, RT-PCR, PI3K activity assays, and immunoprecipitations of PI3K Class IA subunits p85alpha, p85beta, and p110delta from lysed human platelets, as well as Triton X-100-insoluble cytoskeletal preparations from resting and thrombin receptor-activated platelets. We report that p110delta is present in association with p85alpha and p85beta in platelets, both in cytosolic and cytoskeletal fractions. The latter finding is consistent with the proposed role of p110delta in cytoskeletal function.     

PI3K in lymphocyte development,differentiation and activation

Phosphoinositide 3-kinases (PI3Ks) regulate numerous biological processes, including cell growth, differentiation, survival, proliferation, migration and metabolism. In the immune system, impaired PI3K signalling leads to immunodeficiency, whereas unrestrained PI3K signalling contributes to autoimmunity and leukaemia. New insights into the role of PI3Ks in lymphocyte biology have been derived from gene-targeting studies, which have identified the PI3K subunits that are involved in B-cell and T-cell signalling. In particular, the catalytic subunit p110delta seems to be adapted to transmit antigen-receptor signalling in B and T cells. Additional recent work has provided new insights into the molecular interactions that lead to PI3K activation and the signalling pathways that are regulated by PI3K.     

PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane–substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5–10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells.We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.     

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.     

The effect of deleting p110delta on the phenotype and function of PTEN-deficient B cells

Control of the intracellular levels of phosphatidylinositol-(3, 4, 5)-trisphosphate by PI3K and phosphatase and tensin homolog (PTEN) is essential for B cell development and differentiation. Deletion of the PI3K catalytic subunit p110delta leads to a severe reduction in B1 and marginal zone (MZ) B cells, whereas deletion of PTEN results in their expansion. We have examined the relationship between these two molecules by generating mice with a B cell-specific deletion of PTEN (PTENB) and a concurrent germline deletion of p110delta. The expanded B1 cell population of PTENB mice was reduced to normal levels in PTENB/p110delta mutant mice, indicating a critical role for the p110delta isoform in the expansion of B1 cells. However, numbers of MZ B cells in the PTENB/p110delta mutants was intermediate between wild-type and PTENB-deficient mice, suggesting an additional role for other PI3K catalytic isoforms in MZ differentiation. Furthermore, the defective class switch recombination in PTENB B cells was only partially reversed in PTENB/p110delta double mutant B cells. These results demonstrate an epistatic relationship between p110delta and PTEN. In addition, they also suggest that additional PI3K catalytic subunits contribute to B cell development and function.     

Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin- and phosphatidylinositol 3-kinase-dependent PKC alpha activation.

In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and beta(1) integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) alpha is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify beta(1) integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and beta(1) integrin. Moreover, we demonstrate that PKC alpha is constitutively associated with beta(1) integrin. These data are consistent with PKC alpha-dependent exocytosis of activated beta(1) integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKC alpha-driven internalization of beta(1) integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and beta(1) integrin-mediated binding to fibronectin are required for the recruitment and activation of PKC alpha. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin.     

p110beta and p110delta phosphatidylinositol 3-kinases up-regulate Fc(epsilon)RI-activated Ca2+ influx by enhancing inositol 1,4,5-trisphosphate production

Fc(epsilon)RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fc(epsilon)RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha, lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fc(epsilon)RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.     

Identification and characterization of the autophosphorylation sites of phosphoinositide 3-kinase isoforms beta and gamma

Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I(A) PI3K beta and p101-coupled class I(B) PI3K gamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110 beta and serine 1101 of p110 gamma). Like other class I(A) PI3K isoforms, autophosphorylation of p110 beta resulted in down-regulated PI3K beta lipid kinase activity. However, no inhibitory effect of p110 gamma autophosphorylation on PI3K gamma lipid kinase activity was observed. Moreover, PI3K beta and PI3K gamma differed in the regulation of their autophosphorylation. Whereas p110 beta autophosphorylation was stimulated neither by G beta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110 gamma was significantly enhanced by G beta gamma in a time- and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3K beta and PI3K gamma occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3K beta and PI3K gamma.     

Cutting edge: differential roles for phosphoinositide 3-kinases, p110gamma and p110delta, in lymphocyte chemotaxis and homing

Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110gamma-deficient T cells, but not B cells, show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110delta, a major PI3K isoform in lymphocytes. B cells deficient in p110delta showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110delta-deficient B cells revealed diminished homing to Peyer's patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer's patches. These observations establish that p110gamma and p110delta function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.