The phosphatidylinositol 3-kinase/Akt signaling pathway modulates the endocrine differentiation of trophoblast cells

Activation of Lyn, a Src-related nonreceptor tyrosine kinase, in trophoblast cells is associated with trophoblast giant cell differentiation. The purpose of the present work was to use Lyn as a tool to identify signaling pathways regulating the endocrine differentiation of trophoblast cells. The Src homology 3 domain of Lyn was shown to display differentiation-dependent associations with other regulatory proteins, including phosphatidylinositol 3-kinase (PI3-K). PI3-K activation was dependent upon trophoblast giant cell differentiation. The downstream mediator of PI3-K, Akt/protein kinase B, also exhibited differentiation-dependent activation. Lyn is a potential regulator of the PI3-K/Akt signaling pathway, as are receptor tyrosine kinases. Protein tyrosine kinase profiling was used to identify two candidate regulators of the PI3-K/Akt pathway, fibroblast growth factor receptor-1 and Sky. At least part of the activation of Akt in differentiating trophoblast giant cells involves an autocrine growth arrest-specific-6-Sky signaling pathway. Inhibition of PI3-K activities via treatment with LY294002 disrupted Akt activation and interfered with the endocrine differentiation of trophoblast giant cells. In summary, activation of the PI3-K/Akt signaling pathway regulates the development of the differentiated trophoblast giant cell phenotype.     

Lyn contributes to regulation of multiple Kit-dependent signaling pathways in murine bone marrow mast cells

SCF induces autophosphorylation of Kit and activates a variety of signaling components including Jnks, Erks, PI 3 Kinase, the JAK-Stat pathway and members of the Src family. Previously we showed that Lyn is activated at multiple points during SCF-induced cell cycle progression and contributes to SCF-mediated growth, chemotaxis and internalization of Kit. However, the Kit-dependent biochemical events that require Lyn are unknown. In this study, we used Lyn-deficient bone marrow mast cells (BMMC) to examine the contribution of this Src family member to tyrosine phosphorylation of Kit and SCF-induced activation of Jnks, Akt, Stat3 and Erks. Although surface expression of Kit was increased in Lyn-deficient BMMC, SCF-induced phosphorylation and growth was reduced compared to wild-type BMMC. Downstream of Kit, SCF-induced activation of Jnks was markedly reduced in Lyn-deficient BMMC. Further, Lyn was required for SCF-induced tyrosine phosphorylation of Stat3. Interestingly, Kit was constitutively associated with PI 3 Kinase in Lyn-deficient BMMC and this correlated with constitutive phosphorylation of Akt. This was in marked contrast to wild-type BMMC, where both these events were induced by SCF. These data indicate that in BMMC, Lyn contributes to SCF-induced phosphorylation of Kit, as well as phosphorylation of Jnks and Stat3. In contrast, Lyn may negatively regulate the PI 3 Kinase/Akt pathway. The opposing effects of Lyn on these signaling pathways may explain the pleiotropic effects ascribed to this Src family member in the literature.     

Inhibition of the Jun N-terminal protein kinase pathway by SHIP-1, a lipid phosphatase that interacts with the adaptor molecule Dok-3

Dok-3 is a Dok-related adaptor expressed in B cells and macrophages. Previously, we reported that Dok-3 is an inhibitor of B-cell activation in A20 B cells and that it associates with SHIP-1, a 5' inositol-specific lipid phosphatase, as well as Csk, a negative regulator of Src kinases. Here, we demonstrate that Dok-3 suppresses B-cell activation by way of its interaction with SHIP-1, rather than Csk. Our biochemical analyses showed that the Dok-3-SHIP-1 complex acts by selectively inhibiting the B-cell receptor (BCR)-evoked activation of the Jun N-terminal protein kinase (JNK) cascade without affecting overall protein tyrosine phosphorylation or activation of previously described SHIP-1 targets like Btk and Akt/PKB. Studies of B cells derived from SHIP-1-deficient mice showed that BCR-triggered activation of JNK is enhanced in the absence of SHIP-1, implying that the Dok-3-SHIP-1 complex (or a related mechanism) is a physiological negative regulator of the JNK cascade in normal B cells. Together, these data elucidate the mechanism by which Dok-3 inhibits B-cell activation. Furthermore, they provide evidence that SHIP-1 can be a negative regulator of JNK signaling in B cells.     

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells

S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.     

Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.     

Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI

Aggregation of the high affinity receptor for IgE (FcepsilonRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcepsilonRI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with FcepsilonRI beta subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcepsilonRI beta. The latter induced much higher levels of FcepsilonRI beta phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with FcepsilonRI beta, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant FcepsilonRI beta showed that FcepsilonRI beta subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcepsilonRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcepsilonRI beta-ITAM.     

Lyn-dependent signaling regulates the innate immune response by controlling dendritic cell activation of NK cells

The innate immune response is a first line of defense against invading pathogens; however, the magnitude of this response must be tightly regulated, as hyper- or suboptimal responses can be detrimental to the host. Systemic inflammation resulting from bacterial infection can lead to sepsis, which remains a serious problem with high mortality rates. Lyn tyrosine kinase plays a key role in adaptive immunity, although its role in innate immunity remains unclear. In this study, we show that Lyn gain-of-function (Lyn(up/up)) mice display enhanced sensitivity to endotoxin and succumb to upregulated proinflammatory cytokine production at a dose well tolerated by control animals. Endotoxin sensitivity in Lyn(up/up) mice depends on dendritic cells (DCs) and NK cells and occurs though a mechanism involving increased maturation and activation of the DC compartment, leading to elevated production of IFN-γ by NK cells. We further show that modulation of endotoxin-induced signal transduction in DCs by Lyn involves the phosphatases Src homology 2 domain-containing phosphatase-1 and SHIP-1. Collectively, we demonstrate that Lyn regulates DC physiology such that alterations in Lyn-dependent signaling have profound effects on the nature and magnitude of inflammatory responses. Our studies highlight how perturbations in signaling pathways controlling DC/NK cell-regulated responses to microbial products can profoundly affect the magnitude of innate immune responses.     

Suppressor of cytokine signaling 1 interacts with the macrophage colony-stimulating factor receptor and negatively regulates its proliferation signal

Macrophage colony-stimulating factor receptor (M-CSF-R) is a tyrosine kinase that regulates proliferation, differentiation, and cell survival during monocytic lineage development. Upon activation, M-CSF-R dimerizes and autophosphorylates on specific tyrosines, creating binding sites for several cytoplasmic SH2-containing signaling molecules that relay and modulate the M-CSF signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and growth factor signaling pathways. Using the yeast two-hybrid system, in vitro glutathione S-transferase-M-CSF-R pull-down, and in vivo coimmunoprecipitation experiments, we demonstrated a direct interaction between the SH2 domain of Socs1 and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert region. Moreover, Socs1 is tyrosine-phosphorylated in response to M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML hematopoietic cell lines decreased their growth rates in the presence of limiting concentrations of M-CSF. However, Socs1 expression did not totally suppress long term cell growth in the presence of saturating M-CSF concentrations, in contrast to other cytokines such as stem cell factor and interleukin 3. Taken together, these results suggest that Socs1 is an M-CSF-R-binding partner involved in negative regulation of proliferation signaling and that it differentially affects cytokine receptor signals.     

Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection

The mechanism of phagocytosis of pathogens remains to be fully characterized. We report a novel phagocytosis pathway for Pseudomonas aeruginosa, which is initiated by cholesterol-rich membrane rafts and is dependent on Lyn, primarily an immune regulator with both positive and negative roles. Blocking of Lyn or blocking of cholesterol synthesis significantly inhibited phagocytosis by alveolar macrophages. We found that Lyn, via Src homology 2 and 3 domains, bound to and then activated PI3K and Akt to regulate intracellular routing of the engulfed P. aeruginosa. Further analysis indicates that Lyn and raft components entered in phagosomes and late lysosomes. Finally, respiratory burst was dependent on Lyn and membrane rafts, as confirmed by small interfering RNA and dominant-negative strategies. Our investigations demonstrate that Lyn along with membrane rafts plays a fundamental role in phagocytosis by alveolar macrophages during infection.     

CD300F blocks both MyD88 and TRIF-mediated TLR signaling through activation of Src homology region 2 domain-containing phosphatase 1

CD300F is known to exhibit inhibitory activity in myeloid cells through its intracellular ITIM. To investigate the effect of CD300F stimulation on TLR signaling, the human acute monocytic leukemia cell line THP-1 was treated with CD300F-specific mAbs or two synthetic peptides that represented the ITIM-like domains of CD300F. Treatment with these agents blocked TLR2-, 3-, 4-, and 9-mediated expression of proinflammatory mediators such as IL-8 and matrix metalloproteinase-9. The luciferase reporter assay in 293T cells and Western blot analysis of THP-1 cells revealed that these inhibitory actions were effective in pathways involving MyD88 and/or TRIF of TLR signaling and associated with marked suppression of IκB kinase activation, phosphorylation/degradation of IκB, and subsequent activation of NF-κB. Use of specific inhibitors and immunoprecipitation analysis further indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatase-1, a protein tyrosine phosphatase with inhibitory activity in hematopoietic cells. These data indicate that CD300F is an active regulator of TLR-mediated macrophage activation through its association with Src homology 2 domain-containing phosphatase-1 and that the synthetic peptides can be applied for the regulation of immune responses that are induced by TLRs.     

Src homology 2 domain of overexpressed Lyn kinase is responsible for the acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation

We have previously shown that the overexpression of a Src family kinase, Lyn, and its kinase-negative form, LynKN, in a granulocyte progenitor cell line, GM-I62M, accelerates neutrophilic nuclear lobulation when the cells are cultured in the presence of granulocyte colony-stimulating factor. In this study, we investigated the role of the Src homology 2 (SH2) and SH3 domains of Lyn in the accelerated induction of nuclear lobulation. In contrast to wild-type Lyn, the overexpression of its SH2 domain mutant did not induce the accelerated nuclear morphological changes, but the overexpressed SH3 domain mutant had the same effects as wild-type Lyn. Therefore, the SH2 domain of Lyn is responsible for the accelerated induction of neutrophilic nuclear lobulation upon G-CSF stimulation.     

Dysregulation of Src family kinases in mast cells from epilepsy-resistant ASK versus epilepsy-prone EL mice

EL mice have been used as a model of epilepsy, whereas ASK mice are an epilepsy-resistant variant originating from a colony of EL mice. Mast cell-dependent anaphylaxis is easily inducible by stimulation with IgE and Ag in ASK mice, whereas EL mice are resistant to such stimuli. In this study we have characterized mast cells derived from these two strains. ASK mast cells proliferated more vigorously than EL cells in response to IL-3 and stem cell factor. Although ASK mast cells degranulated less vigorously than EL mast cells upon stimulation with IgE and Ag, ASK cells produced and secreted several-fold more TNF-alpha and IL-2 than EL cells. Consistent with the similarities of these ASK and EL mast cell responses with phenotypes of lyn(-/-) and wild-type mast cells, respectively, Lyn activity was reduced in ASK cells. In addition to the impaired Lyn activity, ASK cells just like lyn(-/-) cells exhibited reduced Syk activity, prolonged activation of ERK and JNK, and enhanced activation of Akt. Furthermore, the lipid raft-resident transmembrane adaptor protein Cbp/PAG that associates with Lyn was hypophosphorylated in ASK cells. Importantly, similar to lyn(-/-) cells, Fyn was hyperactivated in ASK cells. Therefore, these results are consistent with the notion that Lyn-dependent phosphorylation of Cbp/PAG negatively regulates Src family kinases. This study also suggests that reduced activity of Lyn, a negative regulator of mast cell activation, underlies the susceptibility of ASK mice to anaphylaxis and implies that dysregulation of Lyn and other Src family kinases contributes to epileptogenesis.     

Role of two adaptor molecules SLP-76 and LAT in the PI3K signaling pathway in activated T cells

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). We also demonstrated that tyrosine phosphorylation either at the 113 and/or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LAT adaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.     

Genetic interdependence of lyn and negative regulators of B cell receptor signaling in autoimmune disease development

Ab-mediated autoimmune disease is multifaceted and may involve many susceptibility loci. The majority of autoimmune patients are thought to have polymorphisms in a number of genes that interact in different combinations to contribute to disease pathogenesis. Studies in mice and humans have implicated the Lyn protein tyrosine kinase as a regulator of Ab-mediated autoimmune disease. To examine whether haploinsufficiency of Lyn gives rise to cellular and clinical manifestations of autoimmune disease, we evaluated the phenotype of Lyn(+/-) mice. We find that their B cell compartment is significantly perturbed, with reduced numbers of marginal zone and transitional stage 2 B cells, expansion of plasma cells, downregulation of surface IgM, and upregulation of costimulatory molecules. Biochemical studies show that Lyn(+/-) B cells have defects in negative regulation of signaling, whereas Lyn(+/-) mice develop IgG autoantibodies and glomerulonephritis with age. Because Lyn has a pivotal role in the activation of inhibitory phosphatases, we generated mice harboring double heterozygous loss-of-function mutations in Lyn and SHP-1 or Lyn and SHIP-1. Partial inactivation of SHP-1 or SHIP-1 amplifies the consequence of Lyn haploinsufficiency, leading to an accelerated development of autoantibodies and disease. Our data also reveal that the BALB/c background is protective against autoimmune-mediated glomerulonephritis, even in the face of high titer autoantibodies, whereas the C57BL/6 background is susceptible. This study demonstrates that Lyn is a haploinsufficient gene in autoimmune disease and importantly shows that quantitative genetic variation in Lyn-regulated pathways can mirror the complete loss of a single critical inhibitory molecule.     

SH2 domain-containing inositol polyphosphate 5'-phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen.

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcepsilonRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5'-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcepsilonRI stimulus.     

A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.