The scaffolding adapter Gab2, via Shp-2, regulates kit-evoked mast cell proliferation by activating the Rac/JNK pathway

The scaffolding adapter Gab2 mediates cell signaling and responses evoked by various extracellular stimuli including several growth factors. Kit, the receptor for stem cell factor (SCF), plays a critical role in the proliferation and differentiation of a variety of cell types, including mast cells. Kit, via Tyr(567) and Tyr(719), activates Src family kinases (SFK) and PI3K respectively, which converge on the activation of a Rac/JNK pathway required for mast cell proliferation. However, how Kit Tyr(567) signals to Rac/JNK is not well understood. By analyzing Gab2(-/-) mast cells, we find that Gab2 is required for SCF-evoked proliferation, activation of Rac/JNK, and Ras. Upon Kit activation in wild-type mast cells, Gab2 becomes tyrosyl-phosphorylated and associates with Kit and Shp-2. Tyr(567), an SFK binding site in Kit, and SFK activity were required for Gab2 tyrosyl phosphorylation and association with Shp-2. By re-expressing Gab2 or a Gab2 mutant that cannot bind Shp-2 in Gab2(-/-) mast cells or acutely by deleting Shp-2 in mast cells, we found that Gab2 requires Shp-2 for SCF-evoked Rac/JNK, Ras activation, and mast cell proliferation. Lastly, by analyzing mast cells from mice with compound Gab2 and Kit Y719F mutations (i.e., Gab2(-/-): KitY719F/Y719F mice), we find that Gab2, acting in a parallel pathway to PI3K from Kit Tyr(719), regulates mast cell proliferation and development in specific tissues. Our data show that Gab2 via Shp-2 is critical for transmitting signals from Kit Tyr(567) to activate the Rac/JNK pathway controlling mast cell proliferation, which likely contributes to mast cell development in specific tissues.     

Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and β1 integrin receptors

Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes^?/?) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated β1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes^?/? BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and β1 integrins to promote cytoskeletal reorganization and motility of mast cells.     

Scaffolding adapter Grb2-associated binder 2 requires Syk to transmit signals from FcepsilonRI

Scaffolding adapter Grb2-associated binder 2 (Gab2) is a key component of FcepsilonRI signaling in mast cells, required for the activation of PI3K. To understand how Gab2 is activated in FcepsilonRI signaling, we asked which protein tyrosine kinase is required for Gab2 phosphorylation. We found that Gab2 tyrosyl phosphorylation requires Lyn and Syk. In agreement with published results, we found that Fyn also contributes to Gab2 tyrosyl phosphorylation. However, Syk activation is defective in Fyn(-/-) mast cells, suggesting that Syk is the proximal kinase responsible for Gab2 tyrosyl phosphorylation. Then, we asked which domains in Gab2 are required for Gab2 tyrosyl phosphorylation. We found that the Grb2-Src homology 3 (SH3) binding sites are required for, whereas the pleckstrin homology (PH) domain contributes to, Gab2 tyrosyl phosphorylation. Using a protein/lipid overlay assay, we determined that the Gab2 PH domain preferentially binds the PI3K lipid products, PI3, 4,5P3 and PI3, 4P2. Furthermore, the Grb2-SH3 binding sites and PH domain binding to PI3K lipid products are required for Gab2 function in FcepsilonRI-evoked degranulation and Akt activation. Our data strongly suggest a model for Gab2 action in FcepsilonRI signaling. The Grb2 SH3 binding sites play a critical role in bringing Gab2 to FcepsilonRI, whereupon Gab2 becomes tyrosyl-phosphorylated in a Syk-dependent fashion. Phosphorylated Gab2 results in recruitment and activation of PI3K, whose lipid products bind the PH domain of Gab2 and acts in positive feedback loop for sustained PI3K recruitment and phosphatidylinositol-3,4,5-trisphosphate production, required for FcepsilonRI-evoked degranulation of mast cells.     

Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells

Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn(-/-) mice (Lyn(-/-) BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn(-/-) BMMCs release more beta-hexosaminidase than wild-type BMMCs following FcepsilonRI cross-linking and show that multiple mast cell responses to FcepsilonRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase Cgamma isoforms, the mobilization of Ca(2+), the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the alpha(4)beta(1) integrin, VLA-4) are slow to initiate in Lyn(-/-) BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn(-/-) BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn(-/-) BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn(-/-) BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcepsilonRI signaling in Lyn(-/-) BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn(-/-) BMMCs may additionally contribute to their altered signaling properties.     

Citreorosein inhibits degranulation and leukotriene C4 generation through suppression of Syk pathway in mast cells

The aim of this study was to evaluate whether citreorosein (CIT), a naturally occurring anthraquinone isolated from Polygoni cuspidati (P. cuspidati) radix, modulates degranulation and 5-lipoxygenase (5-LO)-dependent leukotriene C(4) (LTC(4)) generation in mast cells. Cit suppresses both degranulation and the generation of LTC(4) in a dose-dependent manner in stem cell factor (SCF)-mediated mouse bone marrow-derived mast cells (BMMCs). With regard to its molecular mechanism of action, we investigated the effects of CIT on intracellular signaling and mast cell activation employing BMMCs. Binding of SCF to c-Kit on mast cell membranes induced increases in intrinsic tyrosine kinase Syk activity and activation of multiple downstream events including phosphorylation of phospholipase Cγ (PLCγ), mobilization of intracellular Ca(2+), phosphatidylinositol 3-kinase (PI3K), Akt, MAP kinases (MAPKs), translocation of phospho-phospholipase A(2) (PLA(2)) and 5-LO. The results from the biochemical analysis demonstrate that CIT attenuates degranulation and LTC(4) generation through the suppression of multiple step signaling and would be beneficial for the prevention of allergic inflammation.     

Integrin-induced tyrosine phosphorylation of protein-tyrosine phosphatase-alpha is required for cytoskeletal reorganization and cell migration

Protein-tyrosine phosphatase-alpha (PTPalpha) activates Src family kinases (SFKs) to promote the integrin-stimulated early autophosphorylation of focal adhesion kinase (FAK). We report here that integrin stimulation induces tyrosine phosphorylation of PTPalpha. PTPalpha was dephosphorylated upon fibroblast detachment from the substratum and rephosphorylated when cells were plated on the integrin ligand fibronectin. alpha PTP phosphorylation occurred at Tyr789 and required SFKs (Src or Fyn/Yes), FAK, and an intact cytoskeleton. It also required active PTPalpha or constitutively active Src. These observations indicate that PTPalpha activates SFKs and that the subsequently activated SFK.FAK tyrosine kinase complex in turn phosphorylates PTPalpha. Reintroduction of wild-type PTPalpha or unphosphorylatable PTPalpha(Y789F) (but not inactive PTPalpha) into PTPalpha-null fibroblasts restored defective integrin-induced SFK activation, FAK phosphorylation, and paxillin phosphorylation. PTPalpha(Y789F) and inactive PTPalpha could not rescue delayed actin stress fiber assembly and focal adhesion formation or defective cell migration. This study distinguishes two roles of PTPalpha in integrin signaling: an early role as an activator of SFKs and FAK with no requirement for PTPalpha phosphorylation and a later downstream role in cytoskeleton-associated events for which PTPalpha phosphorylation at Tyr789 is essential.     

Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH(2)-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1(-/-) cells. Consistently, Gab1(-/-) cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.     

SWAP-70 regulates c-kit-induced mast cell activation, cell-cell adhesion, and migration

SWAP-70, an unusual phosphatidylinositol-3-kinase-dependent protein that interacts with the RhoGTPase Rac, is highly expressed in mast cells. Cultured bone marrow mast cells (BMMC) from SWAP-70(-/-) mice are reduced in FcepsilonRI-triggered degranulation. This report describes the hitherto-unknown role of SWAP-70 in c-kit receptor signaling, a key proliferation and differentiation pathway in mast cells. Consistent with the role of Rac in cell motility and regulation of the actin cytoskeleton, mutant cells show abnormal actin rearrangements and are deficient in migration in vitro and in vivo. SWAP-70(-/-) BMMC are impaired in calcium flux, in proper translocation and activity of Akt kinase (required for mast cell activation and survival), and in translocation of Rac1 and Rac2 upon c-kit stimulation. Adhesion to fibronectin is reduced, but homotypic cell association induced through c-kit is strongly increased in SWAP-70(-/-) BMMC. Homotypic association requires extracellular Ca(2+) and depends on the integrin alpha(L)beta(2) (LFA-1). ERK is hyperactivated upon c-kit signaling in adherent and dispersed mutant cells. Together, we suggest that SWAP-70 is an important regulator of specific effector pathways in c-kit signaling, including mast cell activation, migration, and cell adhesion.     

Suppressor of cytokine signaling 1 regulates an endogenous inhibitor of a mast cell protease

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of c-Kit and interleukin-3 (IL-3) receptor signaling. We examined the role of SOCS1 in regulating IL-3-induced cell growth of primary bone marrow-derived mast cells (BMMCs) from SOCS1-/- mice. Instead of showing increased proliferation, SOCS1-deficient BMMCs responded poorly to IL-3 and stem cell factor. SOCS1-/- BMMCs showed increased apoptosis and defective cell cycle entry. We show that the growth retardation of SOCS1-/- BMMCs was due to a cell intrinsic defect. Protein tyrosine phosphorylation following IL-3 stimulation was markedly diminished in SOCS1-/- BMMCs. Intriguingly, JAK2 and STAT5 proteins were selectively diminished in SOCS1-/- BMMCs, which also showed lower molecular mass products of p85 and Vav suggesting proteolytic degradation. Incubation of the SOCS1-/- BMMC lysate with STAT5, p85, and Vav immunoprecipitated from SOCS1+/+ cells directly demonstrated the dysregulated proteolytic activity in SOCS1-/- BMMCs. The proteolytic activity in SOCS1-/- BMMCs was selectively inhibited by phenylmethylsulfonyl fluoride and soybean trypsin inhibitor, suggesting that the protease regulated by SOCS1 is a tryptase. The dysregulated tryptase in SOCS1-/- BMMCs is unlikely to be mMCP6 or mMCP7, because the enzyme activity was not inhibited by Polybrene but was inhibited by normal mouse plasma. SOCS1+/+ BMMC lysate inhibited the proteolytic activity present in SOCS1-/- BMMC lysate, indicating that SOCS1-/- BMMCs lack an endogenous protease inhibitor. These results show that SOCS1 is required for the expression and/or stability of an endogenous protease inhibitor, which protects mast cells from their own proteolytic enzymes.     

Targeted disruption of the tyrosine phosphatase PTPalpha leads to constitutive downregulation of the kinases Src and Fyn.

A role for the receptor-like protein tyrosine phosphatase alpha (PTPalpha) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPalpha enhances the dephosphorylation and activation of Src and Fyn [1] [2] [3]. We have generated mice lacking catalytically active PTPalpha to address the question of whether PTPalpha is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPalpha allele (PTPalpha-/-) and lacking detectable PTPalpha protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPalpha-/- mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPalpha-/- mice. Thus, PTPalpha is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPalpha-/- mouse brain lysates. These may be PTPalpha substrates or downstream signaling proteins. Taken together, the results indicate that PTPalpha has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.     

Increased expression of genes linked to FcepsilonRI Signaling and to cytokine and chemokine production in Lyn-deficient mast cells

Cross-linking the high-affinity IgE receptor, FcepsilonRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn-/-) mice are hyperresponsive to FcepsilonRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn-/- BMMCs in comparison with wild-type (WT) cells. In this study, we compared gene expression profiles between resting and Ag-activated WT and Lyn-/- BMMCs to identify other factors that may contribute to the hyperresponsiveness of the Lyn-/- cells. Among genes implicated in the positive regulation of FcepsilonRI signaling, mRNA for the tyrosine kinase, Fyn, and for several proteins contributing to calcium regulation are more up-regulated following Ag stimulation in Lyn-/- BMMCs than in WT BMMCs. Conversely, mRNA for the low-affinity IgG receptor (FcgammaRIIB), implicated in negative regulation of FcepsilonRI-mediated signaling, is more down-regulated in Ag-stimulated Lyn-/- BMMCs than in WT BMMCs. Genes coding for proinflammatory cytokines and chemokines (IL-4, IL-6, IL-13, CSF, CCL1, CCL3, CCL5, CCL7, CCL9, and MIP1beta) are all more highly expressed in Ag-stimulated Lyn-/- mast cells than in WT cells. These microarray data identify Lyn as a negative regulator in Ag-stimulated BMMCs of the expression of genes linked to FcepsilonRI signaling and also to the response pathways that lead to allergy and asthma.     

Fer and Fps/Fes participate in a Lyn-dependent pathway from FcepsilonRI to platelet-endothelial cell adhesion molecule 1 to limit mast cell activation

Mast cells express the high affinity IgE receptor FcepsilonRI, which upon aggregation by multivalent antigens elicits signals that cause rapid changes within the mast cell and in the surrounding tissue. We previously showed that FcepsilonRI aggregation caused a rapid increase in phosphorylation of both Fer and Fps/Fes kinases in bone marrow-derived mast cells. In this study, we report that FcepsilonRI aggregation leads to increased Fer/Fps kinase activities and that Fer phosphorylation downstream of FcepsilonRI is independent of Syk, Fyn, and Gab2 but requires Lyn. Activated Fer/Fps readily phosphorylate the C terminus of platelet-endothelial cell adhesion molecule 1 (Pecam-1) on immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a non-ITIM residue (Tyr(700)) in vitro and in transfected cells. Mast cells devoid of Fer/Fps kinase activities display a reduction in FcepsilonRI aggregation-induced tyrosine phosphorylation of Pecam-1, with no defects in recruitment of Shp1/Shp2 phosphatases observed. Lyn-deficient mast cells display a dramatic reduction in Pecam-1 phosphorylation at Tyr(685) and a complete loss of Shp2 recruitment, suggesting a role as an initiator kinase for Pecam-1. Consistent with previous studies of Pecam-1-deficient mast cells, we observe an exaggerated degranulation response in mast cells lacking Fer/Fps kinases at low antigen dosages. Thus, Lyn and Fer/Fps kinases cooperate to phosphorylate Pecam-1 and activate Shp1/Shp2 phosphatases that function in part to limit mast cell activation.     

Participation of Gab1 and Gab2 in IL-22-mediated keratinocyte proliferation,migration, and differentiation

Interleukin-22 (IL-22) is one of the key mediators of keratinocyte alterations in psoriasis. IL-22 inhibits keratinocyte differentiation and induces the migration of human keratinocytes. Grb2-associated binder 1 (Gab1) has been shown to mediate epidermal growth factor-induced epidermal growth and differentiation via interaction with the Src homology-2-containing protein-tyrosine phosphatase (Shp2). In this investigation, we explore the role of Gab1 and Gab2 in IL-22-mediated keratinocyte activities. We show that both Gab1 and Gab2 were tyrosine phosphorylated in IL-22-stimulated HaCaT cells and human primary epidermal keratinocytes and contributed to the activation of Extracellular signal regulated kinase 1/2 (Erk1/2) through interaction with Shp2. We further demonstrate that HaCaT cells infected with adenoviruses expressing Shp2-binding-defective Gab1/2 mutants exhibited decreased cell proliferation and migration, as well as increased differentiation. Moreover, similar results were observed in HaCaT cells infected with adenovirus-based small interfering RNAs targeting Gab1 and/or Gab2. Altogether, these data underscore the critical roles of Gab1 and Gab2 in IL-22-mediated HaCaT cell proliferation, migration, and differentiation.     

c-Kit-mediated overlapping and unique functional and biochemical outcomes via diverse signaling pathways

A critical issue in understanding receptor tyrosine kinase signaling is the individual contribution of diverse signaling pathways in regulating cellular growth, survival, and migration. We generated a functionally and biochemically inert c-Kit receptor that lacked the binding sites for seven early signaling pathways. Restoring the Src family kinase (SFK) binding sites in the mutated c-Kit receptor restored cellular survival and migration but only partially rescued proliferation and was associated with the rescue of the Ras/mitogen-activated protein kinase, Rac/JNK kinase, and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt pathways. In contrast, restoring the PI-3 kinase binding site in the mutated receptor did not affect cellular proliferation but resulted in a modest correction in cell survival and migration, despite a complete rescue in the activation of the PI-3 kinase/Akt pathway. Surprisingly, restoring the binding sites for Grb2, Grb7, or phospholipase C-gamma had no effect on cellular growth or survival, migration, or activation of any of the downstream signaling pathways. These results argue that SFKs play a unique role in the control of multiple cellular functions and in the activation of distinct biochemical pathways via c-Kit.