Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. II. Effects of LFM-A13, a specific Btk inhibitor

Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils. LFM-A13 decreased the tyrosine phosphorylation induced by fMet-Leu-Phe and inhibited the production of superoxide anions and the stimulation of adhesion, chemotaxis, and phospholipase D activity. We observed a decreased accumulation of phosphatidylinositol-3,4,5-trisphosphate in response to fMet-Leu-Phe in LFM-A13-pretreated cells even though the inhibitor had no direct effect on the lipid kinase activity of the p110 gamma or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110 gamma by fMet-Leu-Phe. The phosphorylation of Akt and of extracellular signal-regulated kinases 1/2 and p38 were similarly inhibited by LFM-A13. LFM-A13 also negatively affected the translocation of Rac-2, RhoA, ADP ribosylation factor-1, Tec, Bmx, and Btk induced by fMet-Leu-Phe. The results of this study provide evidence for an involvement of Btk and possibly other Tec kinase family members in the regulation of the functional responsiveness of human neutrophils and link these events, in part at least, to the modulation of levels of phosphatidylinositol-3,4,5-trisphosphate.     

Tec kinase signaling in T cells is regulated by phosphatidylinositol 3-kinase and the Tec pleckstrin homology domain

Tec, the prototypical member of the Tec family of tyrosine kinases, is abundantly expressed in T cells and other hemopoietic cell types. Although the functions of Itk and Txk have recently been investigated, little is known about the role of Tec in T cells. Using antisense oligonucleotide treatment to deplete Tec protein from primary T cells, we demonstrate that Tec plays a role in TCR signaling leading to IL-2 gene induction. Interestingly, Tec kinases are the only known family of tyrosine kinases containing a pleckstrin homology (PH) domain. Using several PH domain mutants overexpressed in Jurkat T cells, we show that the Tec PH domain is required for Tec-mediated IL-2 gene induction and TCR-mediated Tec tyrosine phosphorylation. Furthermore, we show that Tec colocalizes with the TCR after TCR cross-linking, and that both the Tec PH and Src homology (SH) 2 domains play a role in this association. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, abolishes Tec-mediated IL-2 gene induction and Tec tyrosine phosphorylation, and partially suppresses Tec colocalization with the activated TCR. Thus, our data implicate the Tec kinase PH domain and phosphatidylinositol 3-kinase in Tec signaling downstream of the TCR.     

Tyrosine phosphorylation of a 34-kDa protein induced by cross-linking a novel glycosylphosphatidylinositol-anchored glycoprotein (GPI-80) on human neutrophils that may regulate their adherence and migration

We previously found a novel glycosylphosphatidylinositol (GPI)-anchored glycoprotein designated GPI-80 that modulates complement receptor 3 integrin-dependent adhesion and in vitro transendothelial migration of neutrophils. In this study, we show that antibody-mediated cross-linking of GPI-80 led to rapid tyrosine phosphorylation mainly of a 34-kDa protein (pp34). Chemical inhibitors, such as genistein, sodium orthovanadate, wortmannin, cytochalasin B, Ro 31-8220, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid inhibited this response, whereas pertussis toxin had no effect. These findings demonstrate that the tyrosine phosphorylation of pp34 by cross-linking GPI-80 in human neutrophils involves tyrosine kinases, tyrosine phosphatases, phosphatidylinositol 3-kinase, cytoskeleton reorganization, protein kinase C, and cytoplasmic calcium, but not heterotrimeric G proteins.     

Phosphatidylinositol 3-kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-1 in c-Kit signaling

Dok-1 is an adaptor protein that is a substrate for Bcr-Abl and other tyrosine protein kinases. The presence of pleckstrin homology and phosphotyrosine binding domains as well as multiple tyrosine phosphorylation sites suggests that Dok-1 is involved in protein-protein and/or protein-lipid interactions. Here we show that stimulation of Mo7 hematopoietic cells with c-Kit ligand (KL) induces phosphatidylinositol (PI) 3-kinase-dependent tyrosine phosphorylation and membrane recruitment of Dok-1. Addition of the K-Ras membrane-targeting motif to Dok-1 generated a constitutively membrane-bound Dok-1 protein whose tyrosine phosphorylation was independent of PI 3-kinase. Membrane localization of Dok-1 was required for its ability to function as a negative regulator of cell proliferation. Additional experiments revealed that Dok-1 associated with the juxtamembrane region and C-terminal tail of c-Kit. Lyn promoted phosphorylation of c-Kit and association of c-Kit and Dok-1. Both Lyn and Tec were capable of phosphorylating Dok-1. However, the use of primary bone marrow mast cells from normal and Lyn-deficient mice demonstrated that Lyn is required for KL-dependent Dok-1 tyrosine phosphorylation. Taken together, these data indicate that activation of PI 3-kinase by KL promotes binding of the Dok pleckstrin homology domain and Dok-1 recruitment to the plasma membrane where Dok-1 is phosphorylated by Src and/or Tec family kinases.     

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.     

Divergent signaling pathways link focal adhesion kinase to mitogen-activated protein kinase cascades. Evidence for a role of paxillin in c-Jun NH(2)-terminal kinase activation.

Stimulation of a number of cell surface receptors, including integrins and G protein-coupled receptors, results in the activation of a non-receptor tyrosine kinase known as focal adhesion kinase (FAK). In turn, this kinase is believed to play a critical role in signaling to intracellular kinase cascades controlling gene expression such as extracellular signal-regulated kinases (ERKs), by a yet poorly defined mechanism. Furthermore, whether this tyrosine kinase also mediates the activation of other mitogen-activated protein kinase family members, such as c-Jun NH(2)-terminal kinases (JNKs), is still unclear. We show here that the activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects were found to be phosphatidylinositol 3-kinase-independent, as FAK effectively stimulated Akt, and wortmannin suppressed Akt but not ERK or JNK activation. As previously reported by others, activation of ERK correlated with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, we provide evidence that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent activation of a biochemical route dependent on small GTP-binding proteins of the Rho family.     

Phosphoinositide 3-kinase: A new effector in signal transduction?

Interest in phosphopinositide 3-kinase (PI 3-kinase) has been fuelled by its identification as a major phosphotyrosyl protein detected in cells following growth factor stimulation and oncogenic transformation. It is found complexed with activated growth factor receptors and non-receptor tyrosine kinases, thus suggesting that it participates in the signal transduction pathways initiated by the activation of tyrosine kinases. PI 3-kinase phosphorylates the 3-position in the inositol ring of the well known inositol phospholipids in vitro giving phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [PtdIns3P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃], respectively. The cellular levels of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ rapidly increase in circumstances where PI 3-kinase becomes complexed with tyrosine kinases. Accumulation of the same lipids also occurs in platelets and neutrophils following stimulation of G-protein linked -thrombin and chemotactic peptide receptors, respectively, leading to speculation that one or both of these lipids is a new second messenger whose function is not yet known. This review brings together recent information on the isolation, characterization and regulation of PI 3-kinase, the cellular occurrence of 3-phosphorylated inositol phospholipids and possible functions of the PI 3-kinase pathway in cell signalling.     

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.     

Stimulation of human neutrophils by chemotactic factors is associated with the activation of phosphatidylinositol 3-kinase gamma

The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) is associated with an increased synthesis of the highly phosphorylated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)). The aims of the present investigation were to determine whether the newly described, G protein-dependent phosphatidylinositol 3-kinase (PI3K), p110gamma, was involved in the responses to chemotactic factors interacting with G protein-coupled receptors. The presence of p110gamma in neutrophils was first established both at the protein and the mRNA level. Stimulation of the cells with fMet-Leu-Phe or interleukin-8 increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. The time course of this effect (threshold within less than 5 s, maximal activation at 10-15 s) was consistent with that of the generation of PtdIns(3,4,5)P(3). Wortmannin, a PI3K inhibitor, abrogated the effects of fMet-Leu-Phe, which were also significantly inhibited by pertussis toxin. Finally, fMet-Leu-Phe also induced a significant translocation of p110gamma to a particulate fraction derived from these cells. These data indicate that p110gamma represent the major PI3K activated by fMet-Leu-Phe and interleukin-8 at very early time points following the stimulation of human neutrophils.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation.     

Tec kinase Itk forms membrane clusters specifically in the vicinity of recruiting receptors

The Tec family of tyrosine kinases transduces signals from antigen and other receptors in cells of the hematopoietic system. In particular, interleukin-2 inducible T cell kinase (Itk) plays an important role in modulating T cell development and activation. Itk is activated by receptors via a phosphatidylinositol 3-kinase-mediated pathway, which results in recruitment of Itk to the plasma membrane via its pleckstrin homology domain. We show here that membrane localization of Itk results in the formation of clusters of at least two molecules within 80 A of each other, which is dependent on the integrity of its pleckstrin homology domain. By contrast, the proline-rich region within the Tec homology domain, SH3 or SH2 domains, or kinase activity were not required for this event. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane as only receptors that recruit phosphatidylinositol 3-kinase reside in the same membrane vicinity as the recruited Itk. Our results indicate that Itk forms dimers in the membrane and that receptors that recruit Itk do so to specific membrane regions.     

Signaling to migration in neutrophils: importance of localized pathways

Neutrophils, a major type of blood leukocytes, are indispensable for host defense of bacterial infections. Directed migration in a gradient of chemotactic stimuli enables these cells to rapidly find the site of infection and destroy the invading pathogens.

Chemotactic factors bind to seven-transmembrane-domain receptors and activate heterotrimeric G-proteins. Downstream of these proteins a complex interrelated signaling network is activated in human neutrophils. Stimulation of phospholipase Cβ results in activation of protein kinase C isoforms and increases in cytosolic calcium. Activation of the enzyme phosphoinositide 3-kinase results in increased production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidyl 3,4-bisphosphate. In addition, small GTP-binding proteins of the Rho family, the mitogen-activated protein kinase cascade, tyrosine kinases and protein phosphatases are activated. The enzyme phosphoinositide 3-kinase and the small cytosolic GTP-binding proteins Rho and Rac emerge as key regulators of neutrophil migration. A steep internal gradient of phosphatidylinositol 3,4,5-trisphosphate, with a high concentration in the leading lamellae, is thought to regulate polarized actin polymerization and formation of protrusions, together with Rac which may be more directly involved in initiating actin reorganization. Rho may regulate localized myosin activation, tail retraction, cell body traction and dynamics of adhesion.

The impact of these different signaling pathways on reversible actin polymerization, development of polarity, reversible adhesion and migration, and the putative targets of these pathways in neutrophils, are reviewed in this article. Insight into mechanisms regulating migration of neutrophils could potentially lead to novel therapeutic strategies for counteracting chronic activation of neutrophils which leads to tissue damage.     

Tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase correlates with high proliferation rates in sublines derived from the Jurkat leukemia

A prominent tyrosine phosphorylated protein of 85 kDa (p85) was detected in highly proliferative sublines derived from the Jurkat T cell leukemia. We undertook a study to characterize the identity of this protein and its possible role in the hyperproliferative phenotypes observed. Using immunoblot and immunoprecipitation techniques, this protein was characterized as the p85 regulatory subunit of phosphatidylinositol 3-kinase. Cell proliferation and p85 tyrosine phosphorylation was not affected by tyrphostin AG-490, an inhibitor of Jak kinases, wortmannin or LY294002, inhibitors of the activity of the catalytic phosphatidylinositol 3-kinase subunit. Herbimycin-A and PPI, inhibitors of src-like protein tyrosine kinases, and genistein, a general tyrosine kinase inhibitor, inhibited p85 tyrosine phosphorylation and induced cell death in the sublines. PD98059, an inhibitor of Mek, inhibited cell growth of the sublines, but not that of the parental cells. It was concluded that tyrosine phosphorylation of p85 is associated with highly proliferative tumoral phenotypes, at least in T cell leukemias, independent of the phosphatidylinositol 3-kinase activity of the catalytic subunit.     

Regulation of protein kinase B

Protein kinase B (PKB) is a member of the second-messenger regulated subfamily of protein kinases implicated in signalling downstream of growth factor and insulin receptor tyrosine kinases and phosphatidylinositol 3-kinase (PI 3-kinase). PKB is activated by phosphorylation in response to mitogens and survival factors. Membrane recruitment driven by lipid second-messengers derived from PI 3-kinase leads to PKB phosphorylation and activation by upstream kinases (PDK1 and an as yet identified protein kinase). Prolonged stimulation with growth factors results in nuclear translocation, providing evidence that PKB activation at the plasma membrane precedes its nuclear translocation and supporting a role for PKB in signalling from receptor tyrosine kinases to the nucleus.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

Identification of an Allosteric Signaling Network within Tec Family Kinases

The Tec family kinases are tyrosine kinases that function primarily in hematopoietic cells. The catalytic activity of the Tec kinases is positively influenced by the regulatory domains outside of the kinase domain. The current lack of a full-length Tec kinase structure leaves a void in our understanding of how these positive regulatory signals are transmitted to the kinase domain. Recently, a conserved structure within kinases, the ‘regulatory spine’, which assembles and disassembles as a kinase switches between its active and inactive states, has been identified. Here, we define the residues that comprise the regulatory spine within Tec kinases. Compared to previously characterized systems, the Tec kinases contain an extended regulatory spine that includes a conserved methionine within the C-helix and a conserved tryptophan within the Src homology 2-kinase linker of Tec kinases. This extended regulatory spine forms a conduit for transmitting the presence of the regulatory domains of Tec kinases to the catalytic domain. We further show that mutation of the gatekeeper residue at the edge of the regulatory spine stabilizes the regulatory spine, resulting in a constitutively active kinase domain. Importantly, the regulatory spine is preassembled in this gatekeeper mutant, rendering phosphorylation on the activation loop unnecessary for its activity. Moreover, we show that the disruption of the conserved electrostatic interaction between Bruton's tyrosine kinase R544 on the activation loop and Bruton's tyrosine kinase E445 on the C-helix also aids in the assembly of the regulatory spine. Thus, the extended regulatory spine is a key structure that is critical for maintaining the activity of Tec kinases.     

Dual phosphorylation of phosphoinositide 3-kinase adaptor Grb2-associated binder 2 is responsible for superoxide formation synergistically stimulated by Fc gamma and formyl-methionyl-leucyl-phenylalanine receptors in differentiated THP-1 cells

The class Ia phosphoinositide (PI) 3-kinase consisting of p110 catalytic and p85 regulatory subunits is activated by Tyr kinase-linked membrane receptors such as FcgammaRII through the association of p85 with the phosphorylated receptors or adaptors. The heterodimeric PI 3-kinase is also activated by G protein-coupled chemotactic fMLP receptors, and activation of the lipid kinase plays an important role in various immune responses, including superoxide formation in neutrophils. Although fMLP-induced superoxide formation is markedly enhanced in FcgammaRII-primed neutrophils, the molecular mechanisms remain poorly characterized. In this study, we identified two Tyr-phosphorylated proteins, c-Cbl (Casitas B-lineage lymphoma) and Grb2-associated binder 2 (Gab2), as PI 3-kinase adaptors that are Tyr phosphorylated upon the stimulation of FcgammaRII in differentiated neutrophil-like THP-1 cells. Interestingly, Gab2 was, but c-Cbl was not, further Ser/Thr phosphorylated by fMLP. Thus, the adaptor Gab2 appeared to be dually phosphorylated at the Ser/Thr and Tyr residues through the two different types of membrane receptors. The Ser/Thr phosphorylation of Gab2 required the activation of extracellular signal-regulated kinase, and fMLP receptor stimulation indeed activated extracellular signal-regulated kinase in the cells. Enhanced superoxide formation in response to Fcgamma and fMLP was markedly attenuated when the Gab2 Ser/Thr phosphorylation was inhibited. These results show the importance of the dual phosphorylation of PI 3-kinase adaptor Gab2 for the enhanced superoxide formation in neutrophil-type cells.     

Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.