Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines. A mechanism for negative signaling by the Lyn tyrosine kinase

The B cell antigen receptor (BCR) is coupled to the mobilization of Ca(2+) by the protein-tyrosine kinase, Syk. Syk, recruited to the clustered BCR, becomes phosphorylated on three tyrosines (Tyr-317, Tyr-342, and Tyr-346) located within the linker region that separates the C-terminal catalytic domain from the N-terminal tandem Src homology 2 domains. Phosphorylation within the linker region can be either activating or inhibitory to Ca(2+) mobilization depending on the sites that are modified. Syk that is not phosphorylated on linker region tyrosines couples the BCR to Ca(2+) mobilization through a phosphoinositide 3-kinase-dependent pathway. The phosphorylation of Tyr-342 and -346 enhances the phosphorylation and activation of phospholipase C-gamma and the early phase of Ca(2+) mobilization via a phosphoinositide 3-kinase-independent pathway. The phosphorylation of Tyr-317 strongly dampens the Ca(2+) signal. In cells that lack the Src family kinase, Lyn, the phosphorylation of the inhibitory Tyr-317 is suppressed leading to elevated production of inositol 1,4,5-trisphosphate and an amplified Ca(2+) signal. This provides a novel mechanism by which Lyn functions as an inhibitor of BCR-stimulated signaling. Thus, Syk and Lyn combine to determine the pathway through which the BCR is coupled to Ca(2+) mobilization as well as the magnitude and duration of the Ca(2+) flux.     

Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells

The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.     

Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.     

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.     

Observing FcepsilonRI signaling from the inside of the mast cell membrane

We have determined the membrane topography of the high-affinity IgE receptor, FcstraightepsilonRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017-1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcepsilonRI and Lyn are distributed as small clusters (2-9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcepsilonRI clusters contain Lyn. In contrast, there is essentially no FcepsilonRI-Syk colocalization in resting cells. 2 min after FcepsilonRI cross-linking, approximately 10% of Lyn colocalizes with small and medium-sized FcepsilonRI clusters (up to 20 gold particles), whereas approximately 16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20-100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcstraightepsilonRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcstraightepsilonRI signaling.     

TOM1L1 is a Lyn substrate involved in FcepsilonRI signaling in mast cells

Protein-tyrosine kinase Lyn and Syk are critical for antigen-receptor-induced signal transduction in mast cells. To identify novel Lyn/Syk substrates, we screened an RBL-2H3 bacterial expression library for proteins that were tyrosine phosphorylated with baculoviral expressed Lyn or Syk. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known substrates such as SLP-76, LAT, and alpha-tubulin. A potential substrate of Lyn identified was the molecule TOM1L1, which has several domains thought to be important for membrane trafficking and protein-protein interactions. Because the function of TOM1L1 is unclear, the rat TOM1L1 full-length cDNA was isolated and used to express the protein in COS-1 and RBL-2H3 mast cells. In COS-1 cells, the co-transfection of TOM1L1 and Lyn, but not Syk, resulted in the tyrosine phosphorylation of TOM1L1. In RBL-2H3 mast cells, the overexpressed TOM1L1 was strongly tyrosine phosphorylated in non-stimulated cells, and this phosphorylation was enhanced by FcepsilonRI aggregation. By subcellular fractionation, wild-type TOM1L1 was mainly in the cytoplasm with a small fraction constitutively associated with the membrane; this association was markedly reduced in deletion mutants lacking several of the protein interaction domains. The overexpression of TOM1L1 enhanced antigen-induced tumor necrosis factor (TNF) alpha generation and release. Both protein interaction domains (VHS and the coiled-coil domains) were required for the increased TNFalpha release, but not the increased TNFalpha generation. These results suggest that TOM1L1 is a novel protein involved in the FcepsilonRI signal transduction for the generation of cytokines.     

FcepsilonRI aggregation promotes survival of connective tissue-like mast cells but not mucosal-like mast cells

Mast cells play a critical role in IgE-dependent immediate hypersensitivity reactions. This is facilitated by their capacity to release inflammatory mediators and to undergo activation-induced survival upon cross-linking of the high-affinity IgE-receptor (FcepsilonRI). Due to their heterogeneity, mast cells can be divided into two major groups: the connective tissue mast cells and the mucosal mast cells. We have previously shown that IL-3-dependent bone marrow-derived mast cells can undergo activation-induced survival that is dependent on the prosurvival gene A1. In this study, we have used two different protocols to develop murine connective tissue-like mast cells (CTLMC) and mucosal-like mast cells (MLMC) to investigate their capacity to survive an allergic reaction in vitro. In this study, we demonstrate that FcepsilonRI stimulation promotes survival of CTLMC but not MLMC. Similarly, a prominent induction of A1 is observed only in CTLMC but not MLMC. MLMC have a higher basal level of the proapoptotic protein Bim compared with CTLMC. These findings demonstrate a difference among mast cell populations in their ability to undergo activation-induced survival after FcepsilonRI stimulation, which might explain the slower turnover of CTMC in IgE-dependent reactions.     

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.     

Phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor-independent B-cell proliferation

The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies, considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certain common B-cell malignancies Syk is activated even in the absence of BCR engagement, suggesting a wider role for this kinase in lymphomagenesis. In this paper, we have profiled molecular differences between BCR-induced and constitutive Syk activation in terms of phosphorylation of regulatory tyrosine residues, downstream signaling properties and capacity to sustain B-cell proliferation. Analysis of primary chronic lymphocytic leukemia B-cells and diffuse large B-cell lymphoma cell lines revealed that constitutive and BCR-induced Syk activation differ with respect to the phosphorylation status of the regulatory tyrosines at positions 352 and 525/526, with only the first site being phosphorylated in the case of constitutive and both sites in the case of BCR-induced Syk activation. Syk phosphorylated only on Y352 is capable of downstream signaling, as evidenced by experiments with a phosphomimetic mutant in which the activation loop tyrosines (YY525/526) were replaced with phenylalanines. However, phosphorylation at YY525/526 was shown to significantly increase the enzymatic activity of Syk and to be required for sustained PLCγ2, Akt and ERK signaling as well as B-cell transformation. These data demonstrate that constitutively active Syk and Syk activated by BCR crosslinking represent separate stages of Syk activation with distinct signaling properties and transforming capacities.     

Distinct phosphoinositide 3-kinases mediate mast cell degranulation in response to G-protein-coupled versus FcepsilonRI receptors

Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110beta and p85/p110delta but not p85/p110alpha are required for antigen-mediated calcium flux in RBL-2H3 cells (Smith, A. J., Surviladze, Z., Gaudet, E. A., Backer, J. M., Mitchell, C. A., and Wilson, B. S. et al., (2001) J. Biol. Chem. 276, 17213-17220). We now examine the role of Class IA PI 3-kinases isoforms in degranulation itself, using a single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110delta and p110beta blocked FcepsilonR1-mediated degranulation in response to FcepsilonRI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110alpha, despite the fact that these antibodies have no effect on antigen-induced calcium flux. These data suggest that p110alpha mediates a calcium-independent signal during degranulation. In contrast, only p110beta was required for enhancement of antigen-stimulated degranulation by adenosine, which augments mast cell-mediated airway inflammation in asthma. Finally, we examined carbachol-stimulated degranulation in RBL2H3 cells stably expressing the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly, carbachol-stimulated degranulation was blocked by antibody-mediated inhibition of the Class III PI 3-kinase hVPS34 or by titration of its product with FYVE domains. Antibodies against Class IA PI 3-kinases had no effect. These data demonstrate: (a) a calcium-independent role for p110alpha in antigen-stimulated degranulation; (b) a requirement for p110beta in adenosine receptor signaling; and (c) a requirement for hVPS34 during M1 muscarinic receptor signaling. Elucidation of the intersections between these distinct pathways will lead to new insights into mast cell degranulation.     

Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells

We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcepsilonRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcepsilonRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcepsilonRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcepsilonRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcepsilonRI-induced mast cell responses.     

Once phosphorylated, tyrosines in carboxyl terminus of protein-tyrosine kinase Syk interact with signaling proteins, including TULA-2, a negative regulator of mast cell degranulation

Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.     

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

Point mutation of a tyrosine in the linker region of Syk results in a gain of function

The protein tyrosine kinase Syk plays an essential role in Fc epsilon RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc epsilon RI-mediated tyrosine phosphorylation of phospholipase C-gamma and a dramatic enhancement of histamine release. The in vivo Fc epsilon RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc epsilon RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.     

Regulation of Ca2+ signaling in mast cells by tyrosine-phosphorylated and unphosphorylated non-T cell activation linker

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.     

Polygoni cuspidati radix inhibits the activation of Syk kinase in mast cells for antiallergic activity

The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 +/- 2.1 microg/ml for RBL-2H3 mast cells and 46 +/- 3.2 microg/m for bone marrow-derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-alpha and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.     

Overcoming the signaling defect of Lyn-sequestering, signal-curtailing FcepsilonRI dimers: aggregated dimers can dissociate from Lyn and form signaling complexes with Syk

Clustering the tetrameric (alphabetagamma(2)) IgE receptor, FcepsilonRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcepsilonRI beta and gamma subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcepsilonRI dimers formed by a particular anti-FcepsilonRI alpha mAb (H10) initiate signaling through Lyn activation and FcepsilonRI subunit phosphorylation, but cause only modest activation of Syk and little Ca(2+) mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcepsilonRI multimers, induced by adding F(ab')(2) of goat anti-mouse IgG to H10-treated cells, support strong Ca(2+) mobilization and secretion. Accompanying the recovery of signaling, H10-FcepsilonRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase Cgamma2. Immunogold electron microscopy showed that H10-FcepsilonRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic "signaling domains" that accumulate FcepsilonRI and Syk in Ag-treated cells. In contrast, H10-FcepsilonRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcepsilonRI beta from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcepsilonRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcepsilonRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.     

Distinct Wnt signaling pathways have opposing roles in appendage regeneration

In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures damaged by injury or disease. This process, termed epimorphic regeneration, involves progenitor cells created through the reprogramming of differentiated cells or through the activation of resident stem cells. Wnt/beta-catenin signaling regulates progenitor cell fate and proliferation during embryonic development and stem cell function in adults, but its functional involvement in epimorphic regeneration has not been addressed. Using transgenic fish lines, we show that Wnt/beta-catenin signaling is activated in the regenerating zebrafish tail fin and is required for formation and subsequent proliferation of the progenitor cells of the blastema. Wnt/beta-catenin signaling appears to act upstream of FGF signaling, which has recently been found to be essential for fin regeneration. Intriguingly, increased Wnt/beta-catenin signaling is sufficient to augment regeneration, as tail fins regenerate faster in fish heterozygous for a loss-of-function mutation in axin1, a negative regulator of the pathway. Likewise, activation of Wnt/beta-catenin signaling by overexpression of wnt8 increases proliferation of progenitor cells in the regenerating fin. By contrast, overexpression of wnt5b (pipetail) reduces expression of Wnt/beta-catenin target genes, impairs proliferation of progenitors and inhibits fin regeneration. Importantly, fin regeneration is accelerated in wnt5b mutant fish. These data suggest that Wnt/beta-catenin signaling promotes regeneration, whereas a distinct pathway activated by wnt5b acts in a negative-feedback loop to limit regeneration.