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An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response

An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response ARTICLE An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response 1 1 2 2 Yan Liu, Minghua Zhu, Keigo Nishida, Toshio Hirano, and Weiguo Zhang Department of Immunology, Duke University Medical Center, Durham, NC 27710 Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan Cross-linking of the Fc𝛆 RI activates the phosphatidyl inositol 3 kinase (PI3K) and mitogen- activated protein kinase pathways. Previous studies demonstrate that Ras guanyl nucleotide- releasing protein (RasGRP)1 is essential in T cell receptor–mediated Ras-Erk activation. Here, we report that RasGRP1 plays an important role in Fc𝛆 RI-mediated PI3K activation and mast cell function. RasGRP1-defi cient mice failed to mount anaphylactic allergic reactions. −/− RasGRP1 mast cells had markedly reduced degranulation and cytokine production. Although Fc𝛆 RI-mediated Erk activation was normal, PI3K activation was diminished. Consequently, activation of Akt, PIP -dependent kinase, and protein kinase C 𝛅 was defective. Expression of a constitutively active form of N-Ras could rescue the degranulation defect and Akt activa- −/− tion. We further demonstrated that RasGRP1 mast cells were defective in granule translo- cation, microtubule formation, and RhoA activation. Our results identifi ed RasGRP1 as an essential regulator of mast cell function. FcεRI-evoked allergic responses are initiated are impaired. The phosphorylation of SLP-76 CORRESPONDENCE Weiguo Zhang: by mast cells through releasing granules con- and PLCγ1/γ2 as well as calcium fl ux are sig- [email protected] taining histamine, β-hexosaminidase, and mast nifi cantly reduced. Consequently, mast cell de- cell–specifi c proteases and by secreting infl am- granulation and cytokine production are severely Abbreviations used: BMMC, −/− bone marrow–derived mast cell; matory cytokines (1). As a member of the immune aff ected in LAT mast cells (4). DAG, diacylglycerol; GEF, receptor superfamily, the FcεRI is composed After FcεRI aggregation, another Src fam- guanine-nucleotide exchange ily kinase, Fyn, is also activated and phosphory- of IgE-binding α subunit and signal- transducing factor; LAT, linker for activation β and γ subunits (2). Upon cross-linking of the lates Gab2. After phosphorylation, Gab2 binds of T cells; MAPK, mitogen- activated protein kinase; PDK1, FcεRI, the Src family protein tyrosine kinase the p85 subunit of phosphatidyl inositol 3 ki- phosphatidyl inositol-3,4,5- −/− Lyn is activated and phosphorylates the immuno- nase (PI3K). Studies using Gab2 mast cells triphosphate–dependent kinase 1; receptor tyrosine-based activation motifs in the show that Gab2 is essential for PI3K activation PI3K, phosphatidyl inositol 3 kinase; PIP , phosphatidyl 3 cytoplasmic domains of the β and γ subunits (6, 7). However, it is not clear how PI3K is inositol-3,4,5-triphosphate; leading to recruitment of Syk. Syk is then activated after recruitment to the plasma mem- PKC, protein kinase C; PLC, a ctivated and further phosphorylates linker for brane by Gab2. Previous data show that in phospholipase C; RasGRP, Ras guanyl nucleotide-releasing activation of T cells (LAT), SLP-76, and several platelet- derived growth factor receptor–mediated protein; RBD, Ras-binding other signaling molecules (3). signaling, GTP-bound Ras is capable of acti- domain; SCF, stem cell factor. vating PI3K by binding to the p110 catalytic Upon phosphorylation, LAT assembles a complex of signaling proteins containing Grb2, subunit (8). After PI3K activation, phosphatidyl Gads, SLP-76, and phospholipase C (PLC)γ1/γ2 inositol-3,4,5-triphosphate (PIP )-dependent (4, 5). Recruitment of Sos to LAT by Grb2 kinase 1 (PDK1) is relocated to the membrane likely leads to activation of the Ras–mitogen- by binding to PIP and activated through in- activated protein kinase (MAPK) pathway. termolecular auto-phosphorylation (9). PDK1 Binding of PLCγ to LAT is necessary for acti- then phosphorylates and activates Akt and vation of PLCγ. PLCγ catalyzes the generation PKCδ. Fyn-defi cient mast cells have impaired of inositol 1,4,5-triphosphate and diacylglycerol PI3K activation and degranulation (6), indicat- (DAG), which are responsible for inducing ing that the Fyn-initiated pathway is important in mast cell degranulation. Recent studies calcium infl ux and protein kinase C (PKC) activation, respectively. In the absence of LAT, show that cytoskeleton rearrangement, which signaling pathways downstream of the FcεRI is regulated by the PI3K pathway, is required JEM © The Rockefeller University Press $15.00 93 Vol. 204, No. 1, January 22, 2007 93–103 www.jem.org/cgi/doi/10.1084/jem.20061598 The Journal of Experimental Medicine for granule translocation and mast cell degranulation (10–12). of the RasGRP family. It is highly expressed in myeloid It has been suggested that degranulation is a two-step cells, including mast cells (20, 21). It is not known whether process: granule translocation to the plasma membrane RasGRP4 functions in mast cells. and granule–plasma membrane fusion. Although granule Despite the fact that RasGRP1 plays an essential role in translocation requires Fyn/Gab2, RhoA, and microtubule TCR-mediated Ras-MAPK activation, whether it functions formation, granule–plasma membrane fusion is calcium in FcεRI-mediated signaling in mast cells has not been dependent (10). explored. In this study, we demonstrate that RasGRP1 plays Ras guanyl nucleotide-releasing proteins (RasGRPs) are an important role in FcεRI-mediated PI3K activation and a family of proteins that contain a DAG-binding C1 domain, mast cell function. a Ras exchange motif domain, two EF-hands, and a guanine- nucleotide exchange factor (GEF) domain (13). After PLCγ RESULTS activation, RasGRPs are recruited to the plasma membrane Expression of RasGRP1 in mast cells by binding to DAG for subsequent activation of Ras family To assess the potential function of RasGRP1 in mast cells proteins. Among the four RasGRP proteins, RasGRP1 has and allergic responses, we fi rst examined whether RasGRP1 been shown to play an important role in TCR signaling and is expressed in mast cells. Bone marrow–derived mast cells T cell development. T cell development is partially blocked (BMMCs) were derived from the bone marrow cells from −/− −/− at the double-positive stage in RasGRP1 mice (14). Thy- WT and RasGRP1 mice. After growing in medium with mocytes from these mice show a complete lack of Erk ac- IL-3 for 3 wk, >99% of cultured cells were c-Kit and + −/− tivation in response to PMA, a DAG analogue, or TCR FcεRI . RasGRP1 cells expressed similar levels of c-Kit cross-linking, demonstrating that RasGRP1 links the TCR and FcεRI as WT cells (Fig. 1 A), suggesting that RasGRP1 to Ras signaling. It has been suggested that RasGRP1 inter- is dispensable for mast cell diff erentiation and maturation acts with and activates Ras in the Golgi instead of the plasma in vitro. These mast cells were used for detection of membrane (15). RasGRP2 is a candidate oncogene causing RasGRP1 protein and subsequent biochemical and functional leukemia and regulates platelet activation (16, 17). RasGRP3 assays. To detect RasGRP1 expression, lysates of WT and −/− is preferentially expressed in B cells. It is phosphorylated after RasGRP1 mast cells were subjected to anti-RasGRP1 −/− BCR stimulation (18). It has been demonstrated in the DT40 immunoprecipitation. Lysates from WT and RasGRP1 cell line that RasGRP3 is required for optimal activation of thymocytes were used as controls. The total lysates and Erk upon BCR cross-linking (19). RasGRP4 is a new member immunoprecipitates were analyzed by an anti-RasGRP1 Figure 1. RasGRP1 in Fc𝛆 RI-mediated degranulation. (A) Expression (C) Effect of RasGRP1 inactivation on FcεRI-mediated degranulation. −/− of c-Kit and FcεRI in WT and RasGRP1 BMMCs. (B) Expression of BMMCs were sensitized with anti-DNP IgE and then stimulated with vari- RasGRP1 and RasGRP4 in mast cells. Mast cell lysates were analyzed by ous concentrations of DNP-HSA for 10 min. Degranulation data were Western blotting with anti-RasGRP1, RasGRP4, Erk2, or HSP70 antibodies expressed as percentages of the released verses total β-hexosaminidase or immunoprecipitated with anti-RasGRP1 antibodies. The numbers activity and refl ect three independent experiments. (D) Thapsigargin- shown are relative quantifi cation of RasGRP1 in thymocytes and BMMCs. induced degranulation. 94 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE −/− Western blot. As shown in Fig. 1 B, RasGRP1 protein was Impaired cytokine production in RasGRP1 BMMCs clearly detected in WT BMMCs, although not as abundant Upon FcεRI cross-linking, mast cells secrete cytokines, such as in thymocytes. We also examined the expression of as IL-6, TNFα, IL-3, and IL-4. These cytokines play an RasGRP4 by Western blotting with anti-RasGRP4 antibodies. important role in the allergic response (22). Next, we exam- −/− ined FcεRI-mediated cytokine production by RasGRP1 As reported previously (20, 21), mast cells expressed high amounts of RasGRP4, which was not detected in thymo- BMMCs. Total RNAs were prepared from mast cells acti- cytes (Fig. 1 B). vated for 1 h and were used in RT-PCR. Amplifi cation of the β-actin transcript indicated that similar amounts of RasGRP1 is required for mast cell degranulation cDNAs were used (Fig. 2 A). Although IL-6 RNA synthesis was Next, we investigated whether RasGRP1 functions in reduced slightly, transcription of TNFα, IL-4, and IL-3 was −/− FcεRI-evoked degranulation. BMMCs were sensitized with signifi cantly decreased in RasGRP1 BMMCs (Fig. 2 A). anti-DNP IgE and stimulated with DNP-HSA at 1, 10, 100, These results were further confi rmed by quantitation of cyto- and 1,000 ng/ml to induce degranulation, which was deter- kines secreted into the culture medium at 8 h after stimulation. mined by measuring the release of β-hexosaminidase. As Although IL-6 production was relatively normal, secretion of −/− TNFα, IL-3, and IL-4 by RasGRP1 cells after stimula- shown in Fig. 1 C, FcεRI-evoked degranulation was mark- −/− edly impaired in RasGRP1 BMMCs. This defect was not tion was signifi cantly reduced (Fig. 2 B). These results indi- due to their inability to degranulate because thapsigargin, cated that RasGRP1 is required for cytokine production a calcium ATPase inhibitor, was capable of inducing upon FcεRI engagement. −/− RasGRP1 BMMCs to degranulate (Fig. 1 D). To deter- −/− −/− mine whether granule formation was normal in RasGRP1 Severely defective systemic anaphylaxis in RasGRP1 mice mast cells, we stained mast cells with toluidine blue. Similar To investigate the impact of RasGRP1 inactivation on al- −/− granules were seen in WT and RasGRP1 cells (not lergic response in vivo, we performed IgE-evoked systemic −/− depicted). Thus, defective signaling after FcεRI engagement anaphylaxis on WT and RasGRP1 mice. Anti-DNP IgE likely caused the diminished degranulation. was intravenously injected into mice 24 h before challenge Figure 2. Effect of RasGRP1 defi ciency on cytokine release and independent experiments with similar results. (C) Effect of RasGRP1 inac- −/− passive systemic anaphylaxis. (A) FcεRI-induced cytokine RNA synthesis tivation on passive systemic anaphylaxis. WT and RasGRP1 mice were −/− in WT and RasGRP1 BMMCs. Sensitized BMMCs were stimulated with sensitized with anti-DNP IgE. Anaphylaxis was induced by injection with 30 ng/ml DNP-HSA for 1 h or left unstimulated before RNA preparation. DNP-HSA. Histamine concentration in the blood was determined by ELISA −/− cDNAs were serially diluted and used in RT-PCR. Data shown are repre- (n = 5 for both WT and RasGRP1 mice). The horizontal bars indicate sentative of two independent experiments with similar results. (B) FcεRI- mean values. (D) Normal numbers of mast cells in the peritoneum, back induced cytokine secretion. Data shown are representative of two skin, and ear dermis. JEM VOL. 204, January 22, 2007 95 by systemic administration of DNP-HSA. At 1.5 min after DNP IgE and activated with DNP-HSA for 0, 2, 5, 10, and challenge, blood was harvested from these mice. The con- 20 min or with PMA for 5 min. Total lysates were prepared centration of histamine released into the blood was then and analyzed by Western blotting with diff erent antibodies as measured to evaluate IgE-initiated allergic response. A dra- indicated in Fig. 3. Tyrosine phosphorylation of proteins was −/− matic reduction (>95%) in histamine release was observed relatively normal in RasGRP1 cells (Fig. 3 A). Because of −/− in RasGRP1 mice compared with that in WT mice the important role of LAT in organizing the signaling com- (Fig. 2 C). plexes downstream of the FcεRI, we examined phosphoryla- To assess whether the severe defect in systemic anaphy- tion of LAT by blotting with an anti-LATpY191 antibody. −/− laxis was due to a developmental defect of mast cells in Normal LAT phosphorylation was detected in RasGRP1 −/− RasGRP1 mice, we determined the distribution of mast BMMCs (Fig. 3 A). Tyrosine phosphorylation of PLCγ1 and −/− −/− cells in RasGRP1 mice. Comparable numbers of mast PLCγ2 was also comparable in WT and RasGRP1 cells were detected in the ear and back skin dermis (Fig. 2 D BMMCs (Fig. 3 B). In agreement with normal phosphoryla- −/− and not depicted) in WT and RasGRP1 mice. Similar per- tion of PLCγ1 and PLCγ2, FcεRI-initiated calcium infl ux −/− centages of mast cells were also detected in peritoneal cavities was intact in RasGRP1 BMMCs (Fig. 3 C). These results using toluidine blue staining and fl ow cytometry analysis. were consistent with previous reports showing that RasGRP1 −/− Normal numbers of mast cells in RasGRP1 mice indi- functions downstream of PLCγ (13, 14). cated that RasGRP1 is not required during mast cell devel- −/− opment and excluded the possibility that the defective MAPK activation in RasGRP1 mast cells anaphylaxis is a consequence of mast cell developmental RasGRP1 has an established role in TCR-mediated Ras-Erk defects. This conclusion was consistent with our biochemical activation. It is also required in PMA (a DAG analogue) data showing that stem cell factor (SCF) or IL-3–mediated - induced Erk activation (14). Because RasGRP1 was expressed −/− Erk and Akt activation was normal in RasGRP1 BMMCs in mast cells, we next examined whether the Ras-Erk path- (not depicted). Therefore, our in vivo and in vitro data way was aff ected in the absence of RasGRP1. Surprisingly, −/− suggested that RasGRP1 plays an important role in FcεRI- in contrast with the defective Erk activation in RasGRP1 initiated mast cell degranulation and systemic anaphylaxis. thymocytes, FcεRI-mediated Erk activation was normal in −/− RasGRP1 BMMCs (Fig. 3 D). These data indicated that −/− Fc𝛆 RI-mediated proximal signaling in RasGRP1 BMMCs RasGRP1 is not required for Ras-Erk activation in the Our data suggested that defective intracellular signaling might FcεRI signaling pathway. It is possible that other Ras guanyl be responsible for the impaired degranulation and anaphy- nucleotide exchange factors, such as Sos, or other members laxis in the absence of RasGRP1. We next investigated of the RasGRP family may be the primary activators of this whether FcεRI-evoked signaling events are aff ected in pathway. Jnk and p38 were previously shown to be important −/− RasGRP1 mast cells. BMMCs were sensitized with anti- for production of certain cytokines, such as IL-6 (23, 24). Figure 3. Fc𝛆 RI-mediated proximal signaling in WT and (B) Tyrosine phosphorylation of PLCγ1 and PLCγ2. (C) FcεRI-induced cal- −/− RasGRP1 BMMCs. (A) Tyrosine phosphorylation of proteins after cium infl ux. Sensitized mast cells were loaded with Indo-1 and stimulated FcεRI engagement. After sensitization with anti-DNP IgE, BMMCs were with DNP-HSA. The fl uorescence emission ratio at 405–495 nm was mon- stimulated with DNA-HSA or PMA (P) for 5 min. Total lysates were blotted itored by fl ow cytometry. (D) Activation of MAPKs. Whole cell lysates were with an anti-phosphotyrosine (pY) or anti-LATpY191 antibody. A similar analyzed by Western blotting with antibodies against Erk, Jnk, and p38 amount of lysates loaded in each lane was indicated by an anti-LAT blot. and their phosphorylated forms. 96 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE We also examined Jnk and p38 activation by Western blotting We examined Gab2 phosphorylation and its interaction with with anti–phospho-Jnk and p38. No diff erences in the activa- p85. Gab2 was phosphorylated normally and interacted with −/− tion of these MAPKs were observed between WT and p85 in RasGRP1 BMMCs (Fig. 4 B), suggesting that −/− RasGRP1 BMMCs (Fig. 3 D). In addition, PMA-induced RasGRP1 likely controls PI3K activation through a mecha- MAPK activation was also normal in the absence of nism other than regulating its membrane recruitment by RasGRP1 (Fig. 3 D). Collectively, these data suggested that Gab2. SHIP acts as a gatekeeper of mast cell degranulation RasGRP1 is not required in FcεRI-mediated MAPK activation. through hydrolyzing PIP (27). To investigate whether de- −/− creased PIP production in RasGRP1 cells was caused by −/− Diminished activation of PI3K pathway increased SHIP activation in RasGRP1 mast cells, we ex- PI3K and its downstream signaling pathway play impor- amined SHIP expression and phosphorylation. No substantial −/− tant roles in mast cell function and development (7, 25, 26). diff erences were seen between WT and RasGRP1 cells Genetic inactivation of the p110δ isoform of PI3K leads to (Fig. 4 C). defective mast cell development and reduced IgE-induced We next investigated the impact of impaired PI3K activa- degranulation and cytokine release (25). We asked whether tion on the phosphorylation of molecules in the PI3K path- RasGRP1 could be involved in the activation of the PI3K way. Akt is recruited to the cell membrane by PIP and pathway in FcεRI-evoked signaling. To assess the eff ect of phosphorylated by PDK1 (28). After activation, Akt phos- RasGRP1 defi ciency on PI3K activation, we fi rst assayed the phorylates GSK3β and inhibits its activity. Akt and GSK3β PI3K activity by determining production of PIP after FcεRI are critical in FcεRI-induced production of TNFα and IL-2 stimulation. P-labeled mast cells were stimulated with by regulating the transcriptional activity of NF-κB, NFAT, DNP-HSA for 0, 2, 5, and 10 min, and lipid fractions were and AP-1 (29). We examined Akt activation by Western extracted and separated by thin layer chromatography. WT blotting with antibodies against Akt phosphorylated at Ser473 BMMCs had a threefold increase of PIP at 2 min after FcεRI (Fig. 4 D) or Thr308 (Fig. 4 E). FcεRI-mediated Akt phos- −/− stimulation; however, RasGRP1 cells only had an in- phorylation at these two residues was signifi cantly reduced in −/− crease of 50% (Fig. 4 A). The residual PI3K activity in RasGRP1 BMMCs. The phosphorylation of GSK3β −/− −/− RasGRP1 BMMCs might be due to the compensation of (Ser9) was also decreased in RasGRP1 BMMCs (Fig. 4 E). RasGRP1 function by other members of the RasGRP These results could explain the reduced cytokine produc- −/− family. A previous study has shown that the membrane re- tion in RasGRP1 cells. Another molecule downstream of cruitment of PI3K by Gab2 is required for its activation (7). PI3K activation is PDK1. Consistent with reduced PIP −/− Figure 4. Impaired PI3K pathway in RasGRP1 BMMCs. (A) Effect with anti-pY, Gab2, and p85 antibodies. (C) FcεRI-induced SHIP1 (Tyr1020) −/− of RasGRP1 defi ciency on FcεRI-induced PIP production in RasGRP1 phosphorylation. Whole cell lysates were blotted with anti-pSHIP BMMCs. Sensitized cells were labeled with [ P]orthophosphate and stim- (Tyr1020) and SHIP antibodies. (D and E) Effect of RasGRP1 defi ciency on ulated with DNP-HSA. Lipids were then extracted and subjected to thin the PI3K pathway. DNP-HSA or PMA (P) -activated mast cell lysates were layer chromatography. Increased PIP production is plotted in the bottom analyzed by Western blotting with antibodies against phosphorylated AKT panel. Data shown were from a representative of three independent (Ser473 and Thr308), PDK1 (Ser241), PKCδ (Thr505), and GSK3β (Ser9). experiments. (B) Gab2 phosphorylation and its association with p85. Lysates The numbers shown were normalized relative intensities for the phospho- were immunoprecipitated with anti-Gab2 followed by Western blotting rylated Akt, PKD1, and PKCδ, respectively. JEM VOL. 204, January 22, 2007 97 production, phosphorylation of PDK1 was also markedly di- Expression of G12V K-Ras slightly enhanced Akt phosphor- −/− minished (Fig. 4 D). As a result of the defective PI3K path- ylation in RasGRP1 cells, whereas expression of G12V way, phosphorylation of PKCδ, which is mediated by PDK1 H-Ras had no eff ect. These Ras proteins were expressed at (30), was signifi cantly diminished (Fig. 4 D). Defective acti- comparable amounts as detected by an antibody against the −/− vation of Akt, PDK1, and PKCδ in RasGRP1 cells could HA epitope tag (Fig. 5 B). Next, we assayed whether the de- not be bypassed by stimulation with PMA (Fig. 4 D), sug- fects in FcεRI-mediated degranulation could be corrected by gesting that RasGRP1 is required for signaling downstream expression of constitutively active Ras. We performed the of DAG production. Collectively, these data indicated that β-hexosaminidase release assay with transduced mast cells. activation of molecules in the PI3K pathway is aff ected by Although G12V N-Ras rescued the defective degranulation −/− RasGRP1 defi ciency. in RasGRP1 BMMCs, G12V K-Ras or H-Ras had little eff ect (Fig. 5 C). These data indicated that N-Ras is the Ras N-Ras mediates regulation of PI3K by RasGRP1 member that is activated by RasGRP1 in mast cells. To examine whether FcεRI-mediated Ras activation was −/− normal, we performed a Ras assay using the GST protein Impaired granule translocation in RasGRP1 BMMCs fused with the Ras-binding domain (RBD) of Raf (GST- Degranulation is thought to be a two-step process. After Raf-RBD). GTP-bound Ras proteins precipitated by GST- granules are translocated to the plasma membrane, they are Raf-RBD were detected by Western blotting with antibodies fused with the plasma membrane to release their contents against H-Ras, N-Ras, and K-Ras, respectively. As shown in (10). To visualize the granule release, we introduced a CD63- Fig. 5 A, the amount of GTP-bound N-Ras precipitated by GFP fusion protein into BMMCs and monitored granule −/− GST-Raf-RBD was reduced in RasGRP1 cells, whereas movement under a confocal microscope as described previ- the amount of GTP-bound K-Ras was similar in WT and ously (10). CD63 is mainly localized in the granules of mast −/− RasGRP1 cells. GTP-bound H-Ras was not detected cells. Before cross-linking with DNP-HSA, mast cell gran- (not depicted). These data indicated that RasGRP1 is likely ules, as revealed by the CD63-GFP fl uorescence, were mostly required for N-Ras activation in FcεRI signaling. in the cytosol. After cross-linking, the majority of granules To determine whether the reduced N-Ras activation was moved close to the plasma membrane in WT BMMCs. In −/− indeed the cause for defective PI3K activation and degranula- contrast, granules in RasGRP1 cells still remained in the tion, we introduced the constitutively activated forms (G12V) cytosol after FcεRI aggregation (Fig. 6 A). After granules are −/− of N-Ras, K-Ras, and H-Ras into RasGRP1 BMMCs fused with the plasma membrane, CD63 can be detected at by retroviral transduction. Stable transductants were selected the cell surface (10). We also examined CD63 surface expres- −/− and expanded. First, we examined whether PI3K activation sion in WT and RasGRP1 cells by staining cells with an could be restored by detecting Akt activation. As shown anti-CD63 antibody followed by FACS analysis. We de- −/− in Fig. 5 B, phosphorylation of Akt in RasGRP1 cells tected an increase of CD63 surface expression on WT cells −/− expressing G12V N-Ras was similar to that in WT cells. but a very minimal increase on RasGRP1 cells (Fig. 6 B). Figure 5. Activation of N-Ras by RasGRP1. (A) Reduced activation of retroviruses expressing constitutively active forms of N-Ras, K-Ras, and −/− −/− N-Ras in RasGRP1 BMMCs. Sensitized WT and RasGRP1 cells were H-Ras were sensitized with anti-DNP IgE and cross-linked with DNP-HSA. activated with DNP-HSA. Lysates were subjected to GST-Raf-RBD precipi- Whole cell lysates were blotted with anti-pAkt, anti-Akt, and anti-HA tation followed by Western blotting with anti–N-Ras and anti–K-Ras antibodies. (C) Effect of a constitutively active Ras on mast cell degranulation. antibodies. (B) Reconstitution of Akt activation by constitutively active Mast cells were transduced as in B. Mast cell degranulation was assayed −/− N-Ras. WT and RasGRP1 BMMCs transduced with empty vector or by measuring the release of β-hexosaminidase. 98 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE Figure 6. Defective granule translocation, microtubule formation, phalloidin (red). (D) Rac1 and RhoA activation. Whole cell lysates were −/− and RhoA activation in RasGRP1 BMMCs. (A) FcεRI-induced gran- subjected to precipitation by GST-PAK-RBD or GST-Rhotekin-RBD beads, −/− ule translocation in WT and RasGRP1 BMMCs. BMMCs expressing respectively. Rac1 and RhoA were detected by Western blotting with an CD63GFP were visualized by confocal microscopy. (B) FcεRI-induced sur- anti-Rac1 or anti-RhoA antibody. Lysates were also blotted with these face CD63 expression analyzed by FACS. (C) FcεRI-induced cytoskeleton antibodies, showing that similar amounts of proteins were used in this rearrangement. Sensitized BMMCs were stimulated with DNP-HSA (+) or assay. One representative of three independent experiments was shown. buffer only (−) and stained with anti–α-tubulin (green) and rhodamine- These data indicated that RasGRP1 is critical for granule pathways (32, 33). We next examined the activation of Rac1 movement in mast cells. Without RasGRP1, mast cells failed and RhoA by using GST-PAK-PBD and GST-Rhotekin to move their granules to the plasma membrane to release RBD fusion proteins to pull down GTP-bound Rac1 their contents. and RhoA. As shown in Fig. 6 D, activation of Rac1 in −/− RasGRP1 cells was similar to that in WT cells. However, −/− −/− Cytoskeleton rearrangement in RasGRP1 BMMCs activation of RhoA was reduced in RasGRP1 cells. These FcεRI engagement induces cytoskeleton rearrangement that results suggested that RasGRP1 plays an important role in includes microtubule formation and disassembly of F-actin FcεRI-mediated RhoA activation. ring. Granule translocation and degranulation require micro- tubule formation (10, 12). To investigate why granules failed DISCUSSION −/− to translocate in RasGRP1 BMMCs, we examined Disruption of RasGRP1 had no eff ect on the proximal sig- formation of microtubule and disassembly of the F-actin naling events after FcεRI engagement. Overall, tyrosine structures by staining BMMCs with anti–α-tubulin and phosphorylation of proteins, phosphorylation of LAT and −/− rhodamine-phalloidin, respectively, before and after FcεRI PLCγ1/γ2, and calcium infl ux were intact in RasGRP1 engagement. Cross-linking of sensitized WT mast cells with mast cells. Calcium infl ux in mast cells is dependent on LAT, DNP-HSA intensifi ed network-like staining (green) of tubu- SLP-76, and PLCγ. LAT or SLP76 defi ciency causes a lin in the cytosol, an indication of the formation of microtu- marked decrease of PLCγ phosphorylation and calcium fl ux bules (Fig. 6 C). In the meantime, F-actin ring, indicated by (4, 34). RasGRP proteins are activated after binding to DAG, phalloidin staining (red), collapsed after stimulation. In acti- a product of PLCγ hydrolysis. Thus, it was not surprising that −/− vated RasGRP1 BMMCs, fl uorescence from anti-tubulin RasGRP1 defi ciency did not aff ect FcεRI-mediated proxi- staining was only seen near the plasma membrane as in un- mal signaling and calcium mobilization. Although PI3K lipid stimulated cells, whereas F-actin disassembly was normal (Fig. products are important for optimal recruitment and activa- 6 C). These results indicated that RasGRP1-mediated signal- tion of PLCγ1 (35), as well as Tec family kinases (36), their ing is required for the formation of microtubules after FcεRI importance in FcεRI-mediated calcium mobilization is not engagement. entirely clear. Mast cells defi cient in Gab2 or Fyn, two criti- Rac and Rho GTPases can regulate cytoskeletal organi- cal molecules in FcεRI-mediated PI3K activation, only have zation in eukaryotic cells (31). Recently, RhoA activation slightly reduced calcium fl ux. It is possible that in mast cells, has been shown to be important for microtubule formation FcεRI-mediated PLCγ activation and calcium mobilization and degranulation in mast cells (10). PI3K and its products are mainly dependent on recruitment of PLCγ to the plasma are required for activation of Rho family proteins in several membrane by LAT. Normal PLCγ phosphorylation and JEM VOL. 204, January 22, 2007 99 −/− calcium mobilization in RasGRP1 mast cells suggested However, the mechanism underlying how PI3K is directly that DAG production is likely normal in these cells. Defec- activated after its localization to membrane has not been tive PI3K activation and mast cell function were less likely clearly characterized in mast cells. Our data suggested that full due to failed DAG production because PMA-induced activa- activation of PI3K after FcεRI engagement requires not only tion of Akt, PKCδ, and PDK1 was still diminished (Fig. 4). membrane recruitment of p85 by Gab2, but also activation RasGRP1 defi ciency aff ects Ras-Erk activation in the by RasGRP1. Our results indicated that RasGRP1 is likely TCR signaling pathway (14), but not in the FcεRI path- involved in PI3K activation through N-Ras. The constitu- way based on our data here. Normal activation of Erk in tively active form of N-Ras could rescue defective Akt ac- −/− RasGRP1 mast cells suggested that Sos or other RasGRP tivation and degranulation. GTP-bound Ras has been shown to interact with the p110 subunit of PI3K and induce its acti- family members might play a major role in FcεRI-mediated Ras-Erk activation. Even though we showed that RasGRP1 vation (8, 39, 40). Effi cient PI3K activation in platelet- was expressed in BMMCs, its abundance was not as high as derived growth factor–induced signaling requires the function in T cells. RasGRP4 is a new member of the RasGRP family of Ras (41). Because RasGRP1 defi ciency did not aff ect and is highly expressed in mast cells (20, 21). C3H/HeJ mice Gab2 phosphorylation and the recruitment of p85, it is likely express a unique isoform of RasGRP4 due to an aberrant that RasGRP1 activates N-Ras, which further induces PI3K splicing. This aberrant splicing produces a dysfunctional pro- activation through its interaction with p110 in FcεRI signal- tein that lacks a DAG-binding domain (C1 domain). These ing (Fig. 7). It is not clear why RasGRP1 is only involved in mice are hyporesponsive to methacholine stimulation via the the activation of N-Ras, not K-Ras or H-Ras. Despite the airway (37), suggesting that RasGRP4 may have an impor- structural similarities among these Ras proteins, their diff er- ential posttranslational modifi cations target them to diff erent tant role in mast cell function, although other genes in these mice can also contribute to the hyporesponsiveness. We microdomains at the membrane or subcellular compartments speculate that RasGRP4 might play a similar role in mast (42, 43). Recent studies suggest that upon T cell activation, cells as RasGRP1 in T cells in Ras-Erk activation. It is also RasGRP1 translocates to the Golgi apparatus to activate Ras possible that FcεRI-mediated Erk activation might be inde- (15). Subsequent studies show that N-Ras is activated upon pendent of RasGRP proteins. In mast cells, Shc is phosphor- engagement of the TCR and PLCγ1 activation, and RasGRP1 ylated upon FcεRI engagement (38). Through binding to may activate N-Ras in the Golgi (44). It remains to be deter- the phosphorylated FcεRI β and γ chains, Shc might recruit mined whether activation of N-Ras by RasGRP1 in mast the Grb2–Sos complex to the plasma membrane to initiate cells occurs in the Golgi or the plasma membrane. Ras-MAPK activation. In contrast with its role in FcεRI-mediated signaling, RasGRP1 is not essential for PI3K and Erk activation in IL-3 Published studies have indicated that PI3K is important in mast cell function (1, 3). Gene inactivation or pharmacologi- or SCF signaling. IL-3 initiates the Jak-Stat pathway, whereas cal inhibition of PI3K impairs FcεRI-mediated degranula- SCF activates the c-Kit receptor tyrosine kinase. Upon phos- tion and cytokine release (25, 26). Recruitment of the p85 phorylation, the IL-3 receptor and c-Kit bind to the Shc– subunit of PI3K by Gab2 is required for its activation (7). Grb2–Sos complex directly to activate the Ras-MAPK pathway Figure 7. A proposed model of RasGRP1 in Fc𝛆 RI-mediated signaling. p110 subunit of PI3K, which binds phosphorylated Gab2, and activates Upon FcεRI cross-linking and PIP hydrolysis, RasGRP1 is recruited to the the PI3K pathway. RasGRP1 also controls RhoA activation indirectly membrane by DAG to activate N-Ras. N-Ras interacts with the catalytic through the PI3K pathway. 100 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE IL-3 for 4 h overnight before stimulation with 20 ng/ml DNP-HSA for the and to p85 to activate the PI3K pathway (45, 46). Why indicated time points in each fi gure. For IL-3 or SCF stimulation, cells RasGRP1 is required for the FcεRI pathway, but not the were starved for 24 h in IMDM medium without IL-3 and stimulated with IL-3– or SCF-mediated pathway, is not clear. It is possible that 20 ng/ml IL-3 or SCF, respectively. IL-3– or SCF-mediated PI3K activation does not require Ras. Mast cell degranulation was performed as described previously (49). For −/− −/− To investigate why RasGRP1 cells failed to degranu- systemic anaphylaxis, WT and RasGRP1 mice were sensitized with anti- late, we analyzed FcεRI-mediated granule translocation and DNP IgE for 24 h and challenged with DNP-HSA for 1.5 min. Blood was collected by cardiac puncture, and histamine concentration in the blood was cytoskeletal rearrangement. Our data indicated that granules determined by ELISA. failed to translocate to the plasma membrane, and the forma- −/− tion of microtubules was defective in RasGRP1 cells after Western blotting. Cells were lysed in RIPA buff er for analysis of whole cell FcεRI engagement. F-actin rearrangement, which is regu- lysates or Brij lysis buff er for immunoprecipitation. For Western blotting, 2+ 2+ lated by Ca and Ca -dependent PKCs (10), was normal in samples were separated by SDS-PAGE and transferred onto nitrocellulose −/− 2+ membranes. After blocking with 1% fi sh gelatin, membranes were incubated RasGRP1 cells. This result was expected because Ca with primary antibodies, washed three times, and probed with either goat fl ux was normal in these cells. Our data showed that RhoA anti–mouse or anti–rabbit Ig conjugated with Alexa Fluor 680 (Invitrogen) or −/− activation was defective in RasGRP1 cells. Vav proteins IRDye 800 (Rockland). Membranes were then visualized and quantifi ed with are GEFs for Rho family GTPases (47) and can be regulated an infrared fl uorescence imaging system (LI-COR Bioscience Odyssey system). by the substrates and products of PI3K (32). Diff erent mem- For most of Western blots, samples were resolved on multiple gels and analyzed bers of the Vav family (Vav1, Vav2, and Vav3) may have dif- by immunoblotting with diff erent antibodies. ferent specifi cities toward diff erent Rho GTPases, such as Numeration of mast cells. Peritoneal fl ushes were stained with toluidine Cdc42, Rac1, and RhoA (48). It is not clear which GEF spe- blue. Percentages of mast cells in the peritoneum were calculated as mast cell −/− cifi cally activates Rac1 and RhoA. In RasGRP1 cells, numbers to total cell numbers in fi ve fi elds under 200× magnifi cation. Rac1 activation was relatively normal, whereas RhoA activa- Skin sections from the back skin and ear dermis were stained with toluidine tion was reduced. It is possible that RasGRP1 activates the blue. Mast cell numbers were counted from at least fi ve sections from −/− each WT (n = 3) and RasGRP1 mouse (n = 3). Data are presented as PI3K pathway, which in turns activates a specifi c GEF mean ± SD. for RhoA, although we cannot exclude the possibility that RasGRP1 is also an exchange factor for RhoA in the FcεRI Calcium infl ux, cytokine production, and PIP measurement. For signaling pathway. calcium infl ux, cells were sensitized with 0.5 μg/ml anti-DNP IgE for 4 h Lyn and Fyn activate two independent pathways upon en- and loaded with 1.5 μM Indo-1 in 1% FBS-HBSS media for 30 min at 30°C. Cells were stimulated with 30 ng/ml DNP-HSA to induce calcium gagement of the FcεRI (6). Activation of the Lyn-Syk-LAT fl ux. The fl uorescence emission ratio at 405–495 nm was monitored by fl ow pathway leads to calcium fl ux and MAPK activation, whereas cytometry. For cytokine production, 2 × 10 anti-DNP IgE sensitized cells the Fyn-Gab2-PI3K pathway is responsible for AKT, PKCδ were stimulated with DNP-HSA for 1 h for RT-PCR and for 8 h for the activation, and degranulation. It has been proposed that there measurement of cytokines released into supernatants using a Bio-Plex cyto- might be cross talks between these two pathways to synergize kine assay (Bio-Rad Laboratories). PIP production was determined using cytokine production and degranulation (6). Our data sug- a protocol described previously (50). gested that RasGRP1 might link LAT phosphorylation and Ras, Rac1, and RhoA activation. For Ras activation, 2 × 10 /ml mast PLCγ activation to PI3K activation, thereby connecting the cells were lysed in a buff er containing 25 mM Hepes, pH 7.5, 150 mM Lyn-initiated pathway to the Fyn-initiated pathway in mast NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mM cells (Fig. 7). In summary, our results indicated that RasGRP1 MgCl , 1 mM EDTA, and 1 mM Na VO . The lysates were incubated with 2 3 4 is an essential regulator of FcεRI-mediated allergic responses. 20 μg GST-Raf-RBD on glutathione beads for 40 min. Beads were washed and boiled in 1× SDS sample buff er. GST-Rhotekin RBD and GST-PAK- MATERIALS AND METHODS PBD fusion protein were provided by K. Burridge (University of North −/− Mice and antibodies. RasGRP1 mice were provided by J.C. Stone Carolina, Chapel Hill, NC). Rac1 and RhoA activation was examined by (University of Alberta, Canada). Mice were housed in specifi c pathogen-free pull-down assays as described previously (51). GTP-bound pan-Ras, H-Ras, conditions, and the use of mice in the experiments described in this study was K-Ras, N-Ras, Rac1, and RhoA were detected by Western blotting with approved by the Duke University Institutional Animal Care Committee. antibodies against each of these proteins, respectively. The following antibodies were used in this study: anti-DNP IgE (SPE-7; Sigma-Aldrich), FcεRIα, and c-Kit (eBioscience); PLCγ2, Erk2, K-Ras, Ras reconstitution by retroviral transduction. Constitutively active forms RhoA, and RasGRP1 (Santa Cruz Biotechnology, Inc.), p85, pTyr (4G10), (G12V) of H-Ras, K-Ras, and N-Ras with an HA tag were subcloned into a retro- PLCγ1, Rac1, and phospho-LATpY191 (Upstate Biotechnology); and pan- viral vector, pMSCV/IRES/Bla. The retroviral plasmids were used to transfect Ras and N-Ras (Calbiochem). AKT, Jnk, p38, PKCδ, PDK1, and other Phoenix cells for retrovirus packaging. 12-d-old BMMCs were transduced with phospho-specifi c antibodies were from Cell Signaling. Anti-LAT (11B12) retroviruses by spin infection. 48 h after transduction, cells were selected with was described previously (49). Anti-RasGRP4 sera were raised by immunizing 5 μg/ml blasticidin and expanded for 10–14 d for subsequent experiments. rabbits with a GST-RasGRP4 fusion protein. Granule translocation and cytoskeletal rearrangement. Examination Mast cell culture, stimulation, and degranulation. Bone marrow cells of granule translocation by confocal microscopy was performed as described −/− were taken from the femurs of WT and RasGRP1 mice and were cul- previously (10). In brief, BMMCs were transduced with pMX-CD63GFP tured in IMDM supplemented with 10% FBS, β-mercaptoethanol, penicillin, retroviruses at day 5 after bone marrow culture in IL-3 medium. At 3 wk and streptomycin in the presence of 5 ng/ml of recombinant IL-3 at 37°C after the initial culture, BMMCs were sensitized with anti-DNP IgE and for 3–6 wk. For stimulation through the FcεRI, 2 × 10 /ml mast cells stimulated with DNP-HSA for 10 min. Cells were immediately fi xed with were sensitized with 0.5 μg/ml anti-DNP IgE in IMDM medium without 4% paraformaldehyde before cytospin and visualized by confocal microscopy. JEM VOL. 204, January 22, 2007 101 For cytoskeletal rearrangement, cells were permeabilized in a buff er con- 16. Dupuy, A.J., K. Morgan, F.C. von Lintig, H. Shen, H. Acar, D.E. taining 0.1% saponin (2% FBS, 1% BSA, and 0.02% sodium azide in PBS) for Hasz, N.A. Jenkins, N.G. Copeland, G.R. Boss, and D.A. Largaespada. 2001. Activation of the Rap1 guanine nucleotide exchange gene, 20 min at room temperature. After cytospin, cells were stained with anti–α- CalDAG-GEF I, in BXH-2 murine myeloid leukemia. J. Biol. Chem. tubulin (Sigma-Aldrich) at 1:50 dilution and incubated with Alexa Fluor 488 276:11804–11811. goat anti–mouse IgG and rhodamine phalloidin (Invitrogen). Confocal mi- 17. Eto, K., R. Murphy, S.W. Kerrigan, A. Bertoni, H. Stuhlmann, T. croscopy was performed using a Zeiss LSM410 confocal system. Nakano, A.D. Leavitt, and S.J. Shattil. 2002. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. −/− We thank Dr. James C. Stone for kindly providing RasGRP1 mice, Ana Sanchez Proc. Natl. Acad. Sci. USA. 99:12819–12824. and Anne Lai for carefully reading the manuscript, and Duke Light Microscopy Core 18. Teixeira, C., S.L. Stang, Y. Zheng, N.S. Beswick, and J.C. Stone. 2003. Facility for confocal microscopy. Integration of DAG signaling systems mediated by PKC-dependent W. Zhang is a scholar of the Leukemia and Lymphoma Society. This work was phosphorylation of RasGRP3. Blood. 102:1414–1420. supported by grants from the National Institutes of Health. 19. Oh-hora, M., S. Johmura, A. Hashimoto, M. Hikida, and T. Kurosaki. The authors have no confl icting fi nancial interests. 2003. Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-γ2 to Ras in B cell receptor signaling. J. Exp. Submitted: 31 July 2006 Med. 198:1841–1851. Accepted: 1 December 2006 20. Reuther, G.W., Q.T. Lambert, J.F. Rebhun, M.A. Caligiuri, L.A. Quilliam, and C.J. Der. 2002. RasGRP4 is a novel Ras activator R E F E R E N C E S isolated from acute myeloid leukemia. J. Biol. Chem. 277:30508–30514. 1. Kinet, J.P. 1999. The high-affi nity IgE receptor (FcεRI): from physiology 21. Yang, Y., L. Li, G.W. Wong, S.A. Krilis, M.S. Madhusudhan, A. Sali, to pathology. Annu. Rev. Immunol. 17:931–972. and R.L. Stevens. 2002. RasGRP4, a new mast cell-restricted Ras 2. Ravetch, J.V. 1994. Fc receptors: rubor redux. Cell. 78:553–560. guanine nucleotide-releasing protein with calcium- and diacylglycerol- 3. Rivera, J., J.R. Cordero, Y. Furumoto, C. Luciano-Montalvo, C. binding motifs. J. Biol. Chem. 277:25756–25774. Gonzalez-Espinosa, M. Kovarova, S. Odom, and V. Parravicini. 2002. 22. Galli, S.J. 2000. Mast cells and basophils. Curr. Opin. Hematol. Macromolecular protein signaling complexes and mast cell responses: 7:32–39. a view of the organization of IgE-dependent mast cell signaling. Mol. 23. Boudreau, R.T., D.W. Hoskin, and T.J. Lin. 2004. Phosphatase in- Immunol. 38:1253–1258. hibition potentiates IL-6 production by mast cells in response to 4. Saitoh, S., R. Arudchandran, T.S. Manetz, W. Zhang, C.L. Sommers, FcεRI-mediated activation: involvement of p38 MAPK. J. Leukoc. Biol. P.E. Love, J. Rivera, and L.E. Samelson. 2000. LAT is essential for 76:1075–1081. FcεRI-mediated mast cell activation. Immunity. 12:525–535. 24. Ishizuka, T., N. Terada, P. Gerwins, E. Hamelmann, A. Oshiba, G.R. 5. Samelson, L.E. 2002. Signal transduction mediated by the T cell an- Fanger, G.L. Johnson, and E.W. Gelfand. 1997. Mast cell tumor necrosis tigen receptor: the role of adapter proteins. Annu. Rev. Immunol. factor alpha production is regulated by MEK kinases. Proc. Natl. Acad. 20:371–394. Sci. USA. 94:6358–6363. 6. Parravicini, V., M. Gadina, M. Kovarova, S. Odom, C. Gonzalez- 25. Ali, K., A. Bilancio, M. Thomas, W. Pearce, A.M. Gilfi llan, C. Tkaczyk, Espinosa, Y. Furumoto, S. Saitoh, L.E. Samelson, J.J. O’Shea, and J. N. Kuehn, A. Gray, J. Giddings, E. Peskett, et al. 2004. Essential role Rivera. 2002. Fyn kinase initiates complementary signals required for for the p110δ phosphoinositide 3-kinase in the allergic response. Nature. IgE-dependent mast cell degranulation. Nat. Immunol. 3:741–748. 431:1007–1011. 7. Gu, H., K. Saito, L.D. Klaman, J. Shen, T. Fleming, Y. Wang, J.C. 26. Barker, S.A., K.K. Caldwell, A. Hall, A.M. Martinez, J.R. Pfeiff er, Pratt, G. Lin, B. Lim, J.P. Kinet, and B.G. Neel. 2001. Essential role for J.M. Oliver, and B.S. Wilson. 1995. Wortmannin blocks lipid and pro- Gab2 in the allergic response. Nature. 412:186–190. tein kinase activities associated with PI 3-kinase and inhibits a subset 8. Rodriguez-Viciana, P., P.H. Warne, R. Dhand, B. Vanhaesebroeck, of responses induced by Fc epsilon R1 cross-linking. Mol. Biol. Cell. I. Gout, M.J. Fry, M.D. Waterfi eld, and J. Downward. 1994. 6:1145–1158. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 27. Huber, M., C.D. Helgason, J.E. Damen, L. Liu, R.K. Humphries, and 370:527–532. G. Krystal. 1998. The src homology 2-containing inositol phosphatase 9. Wick, M.J., F.J. Ramos, H. Chen, M.J. Quon, L.Q. Dong, and F. (SHIP) is the gatekeeper of mast cell degranulation. Proc. Natl. Acad. Sci. Liu. 2003. Mouse 3-phosphoinositide-dependent protein kinase-1 un- USA. 95:11330–11335. dergoes dimerization and trans-phosphorylation in the activation loop. 28. Vanhaesebroeck, B., and D.R. Alessi. 2000. The PI3K-PDK1 connection: J. Biol. Chem. 278:42913–42919. more than just a road to PKB. Biochem. J. 346:561–576. 10. Nishida, K., S. Yamasaki, Y. Ito, K. Kabu, K. Hattori, T. Tezuka, 29. Kitaura, J., K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, H. Nishizumi, D. Kitamura, R. Goitsuka, R.S. Geha, et al. 2005. and T. Kawakami. 2000. Akt-dependent cytokine production in mast FcεRI-mediated mast cell degranulation requires calcium-independent cells. J. Exp. Med. 192:729–740. microtubule-dependent translocation of granules to the plasma membrane. 30. Le Good, J.A., W.H. Ziegler, D.B. Parekh, D.R. Alessi, P. Cohen, J. Cell Biol. 170:115–126. and P.J. Parker. 1998. Protein kinase C isotypes controlled by phos- 11. Martin-Verdeaux, S., I. Pombo, B. Iannascoli, M. Roa, N. Varin-Blank, phoinositide 3-kinase through the protein kinase PDK1. Science. J. Rivera, and U. Blank. 2003. Evidence of a role for Munc18-2 and 281:2042–2045. microtubules in mast cell granule exocytosis. J. Cell Sci. 116:325–334. 31. Etienne-Manneville, S., and A. Hall. 2002. Rho GTPases in cell 12. Smith, A.J., J.R. Pfeiff er, J. Zhang, A.M. Martinez, G.M. Griffi ths, biology. Nature. 420:629–635. and B.S. Wilson. 2003. Microtubule-dependent transport of secretory 32. Han, J., K. Luby-Phelps, B. Das, X. Shu, Y. Xia, R.D. Mosteller, vesicles in RBL-2H3 cells. Traffi c. 4:302–312. U.M. Krishna, J.R. Falck, M.A. White, and D. Broek. 1998. Role of 13. Ebinu, J.O., D.A. Bottorff , E.Y. Chan, S.L. Stang, R.J. Dunn, and J.C. substrates and products of PI 3-kinase in regulating activation of Rac- Stone. 1998. RasGRP, a Ras guanyl nucleotide-releasing protein with related guanosine triphosphatases by Vav. Science. 279:558–560. calcium- and diacylglycerol-binding motifs. Science. 280:1082–1086. 33. Saci, A., and C.L. Carpenter. 2005. RhoA GTPase regulates B cell re- 14. Dower, N.A., S.L. Stang, D.A. Bottorff , J.O. Ebinu, P. Dickie, H.L. ceptor signaling. Mol. Cell. 17:205–214. Ostergaard, and J.C. Stone. 2000. RasGRP is essential for mouse thy- 34. Pivniouk, V.I., T.R. Martin, J.M. Lu-Kuo, H.R. Katz, H.C. Oettgen, mocyte diff erentiation and TCR signaling. Nat. Immunol. 1:317–321. and R.S. Geha. 1999. SLP-76 defi ciency impairs signaling via the high- 15. Bivona, T.G., I. Perez de Castro, I.M. Ahearn, T.M. Grana, V.K. Chiu, affi nity IgE receptor in mast cells. J. Clin. Invest. 103:1737–1743. P.J. Lockyer, P.J. Cullen, A. Pellicer, A.D. Cox, and M.R. Philips. 35. Bae, Y.S., L.G. Cantley, C.S. Chen, S.R. Kim, K.S. Kwon, and S.G. 2003. Phospholipase Cγ activates Ras on the Golgi apparatus by means Rhee. 1998. Activation of phospholipase C-γ by phosphatidylinositol of RasGRP1. Nature. 424:694–698. 3,4,5-trisphosphate. J. Biol. Chem. 273:4465–4469. 102 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE 36. Scharenberg, A.M., O. El-Hillal, D.A. Fruman, L.O. Beitz, Z. Li, 43. Mor, A., and M.R. Philips. 2006. Compartmentalized ras/mapk signaling. S. Lin, I. Gout, L.C. Cantley, D.J. Rawlings, and J.P. Kinet. 1998. Annu. Rev. Immunol. 24:771–800. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase- 44. Perez de Castro, I., T.G. Bivona, M.R. Philips, and A. Pellicer. 2004. dependent calcium signaling pathway: a target for SHIP-mediated in- Ras activation in Jurkat T cells following low-grade stimulation of hibitory signals. EMBO J. 17:1961–1972. the T-cell receptor is specifi c to N-Ras and occurs only on the Golgi 37. Li, L., Y. Yang, G.W. Wong, and R.L. Stevens. 2003. Mast cells in apparatus. Mol. Cell. Biol. 24:3485–3496. airway hyporesponsive C3H/HeJ mice express a unique isoform of the 45. Roskoski, R., Jr. 2005. Signaling by Kit protein-tyrosine kinase–the signaling protein Ras guanine nucleotide releasing protein 4 that is unre- stem cell factor receptor. Biochem. Biophys. Res. Commun. 337:1–13. sponsive to diacylglycerol and phorbol esters. J. Immunol. 171:390–397. 46. Martinez-Moczygemba, M., and D.P. Huston. 2003. Biology of com- 38. Jabril-Cuenod, B., C. Zhang, A.M. Scharenberg, R. Paolini, R. mon β receptor-signaling cytokines: IL-3, IL-5, and GM-CSF. J. Allergy Numerof, M.A. Beaven, and J.-P. Kinet. 1996. Syk-dependent phos- Clin. Immunol. 112:653–665. phorylation of Shc. A potential link between FcεRI and the Ras/ 47. Crespo, P., K.E. Schuebel, A.A. Ostrom, J.S. Gutkind, and X.R. Bustelo. mitogen-activated protein kinase signaling pathway through SOS and 1997. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP ex- Grb2. J. Biol. Chem. 271:16268–16272. change by the vav proto-oncogene product. Nature. 385:169–172. 39. Marte, B.M., P. Rodriguez-Viciana, S. Wennstrom, P.H. Warne, and J. 48. Abe, K., K.L. Rossman, B. Liu, K.D. Ritola, D. Chiang, S.L. Campbell, Downward. 1997. R-Ras can activate the phosphoinositide 3-kinase but K. Burridge, and C.J. Der. 2000. Vav2 is an activator of Cdc42, Rac1, not the MAP kinase arm of the Ras eff ector pathways. Curr. Biol. 7:63–70. and RhoA. J. Biol. Chem. 275:10141–10149. 40. Kodaki, T., R. Woscholski, B. Hallberg, P. Rodriguez-Viciana, J. 49. Zhu, M., Y. Liu, S. Koonpaew, O. Granillo, and W. Zhang. 2004. Downward, and P.J. Parker. 1994. The activation of phosphatidylinositol Positive and negative regulation of FcεRI-mediated signaling by the 3-kinase by Ras. Curr. Biol. 4:798–806. adaptor protein LAB/NTAL. J. Exp. Med. 200:991–1000. 41. Klinghoff er, R.A., B. Duckworth, M. Valius, L. Cantley, and A. 50. Manetz, T.S., C. Gonzalez-Espinosa, R. Arudchandran, S. Xirasagar, Kazlauskas. 1996. Platelet-derived growth factor-dependent activa- V. Tybulewicz, and J. Rivera. 2001. Vav1 regulates phospholipase tion of phosphatidylinositol 3-kinase is regulated by receptor binding Cγ activation and calcium responses in mast cells. Mol. Cell. Biol. of SH2-domain-containing proteins which infl uence Ras activity. Mol. 21:3763–3774. Cell. Biol. 16:5905–5914. 51. Liu, B.P., and K. Burridge. 2000. Vav2 activates Rac1, Cdc42, and 42. Prior, I., and J. Hancock. 2001. Compartmentalization of Ras proteins. RhoA downstream from growth factor receptors but not β1 integrins. J. Cell Sci. 114:1603–1608. Mol. Cell. Biol. 20:7160–7169. JEM VOL. 204, January 22, 2007 103 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response

Liu, Yan; Zhu, Minghua; Nishida, Keigo; Hirano, Toshio; Zhang, Weiguo
The Journal of Experimental Medicine , Volume 204 (1) – Jan 22, 2007
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Abstract

ARTICLE An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response 1 1 2 2 Yan Liu, Minghua Zhu, Keigo Nishida, Toshio Hirano, and Weiguo Zhang Department of Immunology, Duke University Medical Center, Durham, NC 27710 Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan Cross-linking of the Fc𝛆 RI activates the phosphatidyl inositol 3 kinase (PI3K) and mitogen- activated protein kinase pathways. Previous studies demonstrate that Ras guanyl nucleotide- releasing protein (RasGRP)1 is essential in T cell receptor–mediated Ras-Erk activation. Here, we report that RasGRP1 plays an important role in Fc𝛆 RI-mediated PI3K activation and mast cell function. RasGRP1-defi cient mice failed to mount anaphylactic allergic reactions. −/− RasGRP1 mast cells had markedly reduced degranulation and cytokine production. Although Fc𝛆 RI-mediated Erk activation was normal, PI3K activation was diminished. Consequently, activation of Akt, PIP -dependent kinase, and protein kinase C 𝛅 was defective. Expression of a constitutively active form of N-Ras could rescue the degranulation defect and Akt activa- −/− tion. We further demonstrated that RasGRP1 mast cells were defective in granule translo- cation, microtubule formation, and RhoA activation. Our results identifi ed RasGRP1 as an essential regulator of mast cell function. FcεRI-evoked allergic responses are initiated are impaired. The phosphorylation of SLP-76 CORRESPONDENCE Weiguo Zhang: by mast cells through releasing granules con- and PLCγ1/γ2 as well as calcium fl ux are sig- [email protected] taining histamine, β-hexosaminidase, and mast nifi cantly reduced. Consequently, mast cell de- cell–specifi c proteases and by secreting infl am- granulation and cytokine production are severely Abbreviations used: BMMC, −/− bone marrow–derived mast cell; matory cytokines (1). As a member of the immune aff ected in LAT mast cells (4). DAG, diacylglycerol; GEF, receptor superfamily, the FcεRI is composed After FcεRI aggregation, another Src fam- guanine-nucleotide exchange ily kinase, Fyn, is also activated and phosphory- of IgE-binding α subunit and signal- transducing factor; LAT, linker for activation β and γ subunits (2). Upon cross-linking of the lates Gab2. After phosphorylation, Gab2 binds of T cells; MAPK, mitogen- activated protein kinase; PDK1, FcεRI, the Src family protein tyrosine kinase the p85 subunit of phosphatidyl inositol 3 ki- phosphatidyl inositol-3,4,5- −/− Lyn is activated and phosphorylates the immuno- nase (PI3K). Studies using Gab2 mast cells triphosphate–dependent kinase 1; receptor tyrosine-based activation motifs in the show that Gab2 is essential for PI3K activation PI3K, phosphatidyl inositol 3 kinase; PIP , phosphatidyl 3 cytoplasmic domains of the β and γ subunits (6, 7). However, it is not clear how PI3K is inositol-3,4,5-triphosphate; leading to recruitment of Syk. Syk is then activated after recruitment to the plasma mem- PKC, protein kinase C; PLC, a ctivated and further phosphorylates linker for brane by Gab2. Previous data show that in phospholipase C; RasGRP, Ras guanyl nucleotide-releasing activation of T cells (LAT), SLP-76, and several platelet- derived growth factor receptor–mediated protein; RBD, Ras-binding other signaling molecules (3). signaling, GTP-bound Ras is capable of acti- domain; SCF, stem cell factor. vating PI3K by binding to the p110 catalytic Upon phosphorylation, LAT assembles a complex of signaling proteins containing Grb2, subunit (8). After PI3K activation, phosphatidyl Gads, SLP-76, and phospholipase C (PLC)γ1/γ2 inositol-3,4,5-triphosphate (PIP )-dependent (4, 5). Recruitment of Sos to LAT by Grb2 kinase 1 (PDK1) is relocated to the membrane likely leads to activation of the Ras–mitogen- by binding to PIP and activated through in- activated protein kinase (MAPK) pathway. termolecular auto-phosphorylation (9). PDK1 Binding of PLCγ to LAT is necessary for acti- then phosphorylates and activates Akt and vation of PLCγ. PLCγ catalyzes the generation PKCδ. Fyn-defi cient mast cells have impaired of inositol 1,4,5-triphosphate and diacylglycerol PI3K activation and degranulation (6), indicat- (DAG), which are responsible for inducing ing that the Fyn-initiated pathway is important in mast cell degranulation. Recent studies calcium infl ux and protein kinase C (PKC) activation, respectively. In the absence of LAT, show that cytoskeleton rearrangement, which signaling pathways downstream of the FcεRI is regulated by the PI3K pathway, is required JEM © The Rockefeller University Press $15.00 93 Vol. 204, No. 1, January 22, 2007 93–103 www.jem.org/cgi/doi/10.1084/jem.20061598 The Journal of Experimental Medicine for granule translocation and mast cell degranulation (10–12). of the RasGRP family. It is highly expressed in myeloid It has been suggested that degranulation is a two-step cells, including mast cells (20, 21). It is not known whether process: granule translocation to the plasma membrane RasGRP4 functions in mast cells. and granule–plasma membrane fusion. Although granule Despite the fact that RasGRP1 plays an essential role in translocation requires Fyn/Gab2, RhoA, and microtubule TCR-mediated Ras-MAPK activation, whether it functions formation, granule–plasma membrane fusion is calcium in FcεRI-mediated signaling in mast cells has not been dependent (10). explored. In this study, we demonstrate that RasGRP1 plays Ras guanyl nucleotide-releasing proteins (RasGRPs) are an important role in FcεRI-mediated PI3K activation and a family of proteins that contain a DAG-binding C1 domain, mast cell function. a Ras exchange motif domain, two EF-hands, and a guanine- nucleotide exchange factor (GEF) domain (13). After PLCγ RESULTS activation, RasGRPs are recruited to the plasma membrane Expression of RasGRP1 in mast cells by binding to DAG for subsequent activation of Ras family To assess the potential function of RasGRP1 in mast cells proteins. Among the four RasGRP proteins, RasGRP1 has and allergic responses, we fi rst examined whether RasGRP1 been shown to play an important role in TCR signaling and is expressed in mast cells. Bone marrow–derived mast cells T cell development. T cell development is partially blocked (BMMCs) were derived from the bone marrow cells from −/− −/− at the double-positive stage in RasGRP1 mice (14). Thy- WT and RasGRP1 mice. After growing in medium with mocytes from these mice show a complete lack of Erk ac- IL-3 for 3 wk, >99% of cultured cells were c-Kit and + −/− tivation in response to PMA, a DAG analogue, or TCR FcεRI . RasGRP1 cells expressed similar levels of c-Kit cross-linking, demonstrating that RasGRP1 links the TCR and FcεRI as WT cells (Fig. 1 A), suggesting that RasGRP1 to Ras signaling. It has been suggested that RasGRP1 inter- is dispensable for mast cell diff erentiation and maturation acts with and activates Ras in the Golgi instead of the plasma in vitro. These mast cells were used for detection of membrane (15). RasGRP2 is a candidate oncogene causing RasGRP1 protein and subsequent biochemical and functional leukemia and regulates platelet activation (16, 17). RasGRP3 assays. To detect RasGRP1 expression, lysates of WT and −/− is preferentially expressed in B cells. It is phosphorylated after RasGRP1 mast cells were subjected to anti-RasGRP1 −/− BCR stimulation (18). It has been demonstrated in the DT40 immunoprecipitation. Lysates from WT and RasGRP1 cell line that RasGRP3 is required for optimal activation of thymocytes were used as controls. The total lysates and Erk upon BCR cross-linking (19). RasGRP4 is a new member immunoprecipitates were analyzed by an anti-RasGRP1 Figure 1. RasGRP1 in Fc𝛆 RI-mediated degranulation. (A) Expression (C) Effect of RasGRP1 inactivation on FcεRI-mediated degranulation. −/− of c-Kit and FcεRI in WT and RasGRP1 BMMCs. (B) Expression of BMMCs were sensitized with anti-DNP IgE and then stimulated with vari- RasGRP1 and RasGRP4 in mast cells. Mast cell lysates were analyzed by ous concentrations of DNP-HSA for 10 min. Degranulation data were Western blotting with anti-RasGRP1, RasGRP4, Erk2, or HSP70 antibodies expressed as percentages of the released verses total β-hexosaminidase or immunoprecipitated with anti-RasGRP1 antibodies. The numbers activity and refl ect three independent experiments. (D) Thapsigargin- shown are relative quantifi cation of RasGRP1 in thymocytes and BMMCs. induced degranulation. 94 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE −/− Western blot. As shown in Fig. 1 B, RasGRP1 protein was Impaired cytokine production in RasGRP1 BMMCs clearly detected in WT BMMCs, although not as abundant Upon FcεRI cross-linking, mast cells secrete cytokines, such as in thymocytes. We also examined the expression of as IL-6, TNFα, IL-3, and IL-4. These cytokines play an RasGRP4 by Western blotting with anti-RasGRP4 antibodies. important role in the allergic response (22). Next, we exam- −/− ined FcεRI-mediated cytokine production by RasGRP1 As reported previously (20, 21), mast cells expressed high amounts of RasGRP4, which was not detected in thymo- BMMCs. Total RNAs were prepared from mast cells acti- cytes (Fig. 1 B). vated for 1 h and were used in RT-PCR. Amplifi cation of the β-actin transcript indicated that similar amounts of RasGRP1 is required for mast cell degranulation cDNAs were used (Fig. 2 A). Although IL-6 RNA synthesis was Next, we investigated whether RasGRP1 functions in reduced slightly, transcription of TNFα, IL-4, and IL-3 was −/− FcεRI-evoked degranulation. BMMCs were sensitized with signifi cantly decreased in RasGRP1 BMMCs (Fig. 2 A). anti-DNP IgE and stimulated with DNP-HSA at 1, 10, 100, These results were further confi rmed by quantitation of cyto- and 1,000 ng/ml to induce degranulation, which was deter- kines secreted into the culture medium at 8 h after stimulation. mined by measuring the release of β-hexosaminidase. As Although IL-6 production was relatively normal, secretion of −/− TNFα, IL-3, and IL-4 by RasGRP1 cells after stimula- shown in Fig. 1 C, FcεRI-evoked degranulation was mark- −/− edly impaired in RasGRP1 BMMCs. This defect was not tion was signifi cantly reduced (Fig. 2 B). These results indi- due to their inability to degranulate because thapsigargin, cated that RasGRP1 is required for cytokine production a calcium ATPase inhibitor, was capable of inducing upon FcεRI engagement. −/− RasGRP1 BMMCs to degranulate (Fig. 1 D). To deter- −/− −/− mine whether granule formation was normal in RasGRP1 Severely defective systemic anaphylaxis in RasGRP1 mice mast cells, we stained mast cells with toluidine blue. Similar To investigate the impact of RasGRP1 inactivation on al- −/− granules were seen in WT and RasGRP1 cells (not lergic response in vivo, we performed IgE-evoked systemic −/− depicted). Thus, defective signaling after FcεRI engagement anaphylaxis on WT and RasGRP1 mice. Anti-DNP IgE likely caused the diminished degranulation. was intravenously injected into mice 24 h before challenge Figure 2. Effect of RasGRP1 defi ciency on cytokine release and independent experiments with similar results. (C) Effect of RasGRP1 inac- −/− passive systemic anaphylaxis. (A) FcεRI-induced cytokine RNA synthesis tivation on passive systemic anaphylaxis. WT and RasGRP1 mice were −/− in WT and RasGRP1 BMMCs. Sensitized BMMCs were stimulated with sensitized with anti-DNP IgE. Anaphylaxis was induced by injection with 30 ng/ml DNP-HSA for 1 h or left unstimulated before RNA preparation. DNP-HSA. Histamine concentration in the blood was determined by ELISA −/− cDNAs were serially diluted and used in RT-PCR. Data shown are repre- (n = 5 for both WT and RasGRP1 mice). The horizontal bars indicate sentative of two independent experiments with similar results. (B) FcεRI- mean values. (D) Normal numbers of mast cells in the peritoneum, back induced cytokine secretion. Data shown are representative of two skin, and ear dermis. JEM VOL. 204, January 22, 2007 95 by systemic administration of DNP-HSA. At 1.5 min after DNP IgE and activated with DNP-HSA for 0, 2, 5, 10, and challenge, blood was harvested from these mice. The con- 20 min or with PMA for 5 min. Total lysates were prepared centration of histamine released into the blood was then and analyzed by Western blotting with diff erent antibodies as measured to evaluate IgE-initiated allergic response. A dra- indicated in Fig. 3. Tyrosine phosphorylation of proteins was −/− matic reduction (>95%) in histamine release was observed relatively normal in RasGRP1 cells (Fig. 3 A). Because of −/− in RasGRP1 mice compared with that in WT mice the important role of LAT in organizing the signaling com- (Fig. 2 C). plexes downstream of the FcεRI, we examined phosphoryla- To assess whether the severe defect in systemic anaphy- tion of LAT by blotting with an anti-LATpY191 antibody. −/− laxis was due to a developmental defect of mast cells in Normal LAT phosphorylation was detected in RasGRP1 −/− RasGRP1 mice, we determined the distribution of mast BMMCs (Fig. 3 A). Tyrosine phosphorylation of PLCγ1 and −/− −/− cells in RasGRP1 mice. Comparable numbers of mast PLCγ2 was also comparable in WT and RasGRP1 cells were detected in the ear and back skin dermis (Fig. 2 D BMMCs (Fig. 3 B). In agreement with normal phosphoryla- −/− and not depicted) in WT and RasGRP1 mice. Similar per- tion of PLCγ1 and PLCγ2, FcεRI-initiated calcium infl ux −/− centages of mast cells were also detected in peritoneal cavities was intact in RasGRP1 BMMCs (Fig. 3 C). These results using toluidine blue staining and fl ow cytometry analysis. were consistent with previous reports showing that RasGRP1 −/− Normal numbers of mast cells in RasGRP1 mice indi- functions downstream of PLCγ (13, 14). cated that RasGRP1 is not required during mast cell devel- −/− opment and excluded the possibility that the defective MAPK activation in RasGRP1 mast cells anaphylaxis is a consequence of mast cell developmental RasGRP1 has an established role in TCR-mediated Ras-Erk defects. This conclusion was consistent with our biochemical activation. It is also required in PMA (a DAG analogue) data showing that stem cell factor (SCF) or IL-3–mediated - induced Erk activation (14). Because RasGRP1 was expressed −/− Erk and Akt activation was normal in RasGRP1 BMMCs in mast cells, we next examined whether the Ras-Erk path- (not depicted). Therefore, our in vivo and in vitro data way was aff ected in the absence of RasGRP1. Surprisingly, −/− suggested that RasGRP1 plays an important role in FcεRI- in contrast with the defective Erk activation in RasGRP1 initiated mast cell degranulation and systemic anaphylaxis. thymocytes, FcεRI-mediated Erk activation was normal in −/− RasGRP1 BMMCs (Fig. 3 D). These data indicated that −/− Fc𝛆 RI-mediated proximal signaling in RasGRP1 BMMCs RasGRP1 is not required for Ras-Erk activation in the Our data suggested that defective intracellular signaling might FcεRI signaling pathway. It is possible that other Ras guanyl be responsible for the impaired degranulation and anaphy- nucleotide exchange factors, such as Sos, or other members laxis in the absence of RasGRP1. We next investigated of the RasGRP family may be the primary activators of this whether FcεRI-evoked signaling events are aff ected in pathway. Jnk and p38 were previously shown to be important −/− RasGRP1 mast cells. BMMCs were sensitized with anti- for production of certain cytokines, such as IL-6 (23, 24). Figure 3. Fc𝛆 RI-mediated proximal signaling in WT and (B) Tyrosine phosphorylation of PLCγ1 and PLCγ2. (C) FcεRI-induced cal- −/− RasGRP1 BMMCs. (A) Tyrosine phosphorylation of proteins after cium infl ux. Sensitized mast cells were loaded with Indo-1 and stimulated FcεRI engagement. After sensitization with anti-DNP IgE, BMMCs were with DNP-HSA. The fl uorescence emission ratio at 405–495 nm was mon- stimulated with DNA-HSA or PMA (P) for 5 min. Total lysates were blotted itored by fl ow cytometry. (D) Activation of MAPKs. Whole cell lysates were with an anti-phosphotyrosine (pY) or anti-LATpY191 antibody. A similar analyzed by Western blotting with antibodies against Erk, Jnk, and p38 amount of lysates loaded in each lane was indicated by an anti-LAT blot. and their phosphorylated forms. 96 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE We also examined Jnk and p38 activation by Western blotting We examined Gab2 phosphorylation and its interaction with with anti–phospho-Jnk and p38. No diff erences in the activa- p85. Gab2 was phosphorylated normally and interacted with −/− tion of these MAPKs were observed between WT and p85 in RasGRP1 BMMCs (Fig. 4 B), suggesting that −/− RasGRP1 BMMCs (Fig. 3 D). In addition, PMA-induced RasGRP1 likely controls PI3K activation through a mecha- MAPK activation was also normal in the absence of nism other than regulating its membrane recruitment by RasGRP1 (Fig. 3 D). Collectively, these data suggested that Gab2. SHIP acts as a gatekeeper of mast cell degranulation RasGRP1 is not required in FcεRI-mediated MAPK activation. through hydrolyzing PIP (27). To investigate whether de- −/− creased PIP production in RasGRP1 cells was caused by −/− Diminished activation of PI3K pathway increased SHIP activation in RasGRP1 mast cells, we ex- PI3K and its downstream signaling pathway play impor- amined SHIP expression and phosphorylation. No substantial −/− tant roles in mast cell function and development (7, 25, 26). diff erences were seen between WT and RasGRP1 cells Genetic inactivation of the p110δ isoform of PI3K leads to (Fig. 4 C). defective mast cell development and reduced IgE-induced We next investigated the impact of impaired PI3K activa- degranulation and cytokine release (25). We asked whether tion on the phosphorylation of molecules in the PI3K path- RasGRP1 could be involved in the activation of the PI3K way. Akt is recruited to the cell membrane by PIP and pathway in FcεRI-evoked signaling. To assess the eff ect of phosphorylated by PDK1 (28). After activation, Akt phos- RasGRP1 defi ciency on PI3K activation, we fi rst assayed the phorylates GSK3β and inhibits its activity. Akt and GSK3β PI3K activity by determining production of PIP after FcεRI are critical in FcεRI-induced production of TNFα and IL-2 stimulation. P-labeled mast cells were stimulated with by regulating the transcriptional activity of NF-κB, NFAT, DNP-HSA for 0, 2, 5, and 10 min, and lipid fractions were and AP-1 (29). We examined Akt activation by Western extracted and separated by thin layer chromatography. WT blotting with antibodies against Akt phosphorylated at Ser473 BMMCs had a threefold increase of PIP at 2 min after FcεRI (Fig. 4 D) or Thr308 (Fig. 4 E). FcεRI-mediated Akt phos- −/− stimulation; however, RasGRP1 cells only had an in- phorylation at these two residues was signifi cantly reduced in −/− crease of 50% (Fig. 4 A). The residual PI3K activity in RasGRP1 BMMCs. The phosphorylation of GSK3β −/− −/− RasGRP1 BMMCs might be due to the compensation of (Ser9) was also decreased in RasGRP1 BMMCs (Fig. 4 E). RasGRP1 function by other members of the RasGRP These results could explain the reduced cytokine produc- −/− family. A previous study has shown that the membrane re- tion in RasGRP1 cells. Another molecule downstream of cruitment of PI3K by Gab2 is required for its activation (7). PI3K activation is PDK1. Consistent with reduced PIP −/− Figure 4. Impaired PI3K pathway in RasGRP1 BMMCs. (A) Effect with anti-pY, Gab2, and p85 antibodies. (C) FcεRI-induced SHIP1 (Tyr1020) −/− of RasGRP1 defi ciency on FcεRI-induced PIP production in RasGRP1 phosphorylation. Whole cell lysates were blotted with anti-pSHIP BMMCs. Sensitized cells were labeled with [ P]orthophosphate and stim- (Tyr1020) and SHIP antibodies. (D and E) Effect of RasGRP1 defi ciency on ulated with DNP-HSA. Lipids were then extracted and subjected to thin the PI3K pathway. DNP-HSA or PMA (P) -activated mast cell lysates were layer chromatography. Increased PIP production is plotted in the bottom analyzed by Western blotting with antibodies against phosphorylated AKT panel. Data shown were from a representative of three independent (Ser473 and Thr308), PDK1 (Ser241), PKCδ (Thr505), and GSK3β (Ser9). experiments. (B) Gab2 phosphorylation and its association with p85. Lysates The numbers shown were normalized relative intensities for the phospho- were immunoprecipitated with anti-Gab2 followed by Western blotting rylated Akt, PKD1, and PKCδ, respectively. JEM VOL. 204, January 22, 2007 97 production, phosphorylation of PDK1 was also markedly di- Expression of G12V K-Ras slightly enhanced Akt phosphor- −/− minished (Fig. 4 D). As a result of the defective PI3K path- ylation in RasGRP1 cells, whereas expression of G12V way, phosphorylation of PKCδ, which is mediated by PDK1 H-Ras had no eff ect. These Ras proteins were expressed at (30), was signifi cantly diminished (Fig. 4 D). Defective acti- comparable amounts as detected by an antibody against the −/− vation of Akt, PDK1, and PKCδ in RasGRP1 cells could HA epitope tag (Fig. 5 B). Next, we assayed whether the de- not be bypassed by stimulation with PMA (Fig. 4 D), sug- fects in FcεRI-mediated degranulation could be corrected by gesting that RasGRP1 is required for signaling downstream expression of constitutively active Ras. We performed the of DAG production. Collectively, these data indicated that β-hexosaminidase release assay with transduced mast cells. activation of molecules in the PI3K pathway is aff ected by Although G12V N-Ras rescued the defective degranulation −/− RasGRP1 defi ciency. in RasGRP1 BMMCs, G12V K-Ras or H-Ras had little eff ect (Fig. 5 C). These data indicated that N-Ras is the Ras N-Ras mediates regulation of PI3K by RasGRP1 member that is activated by RasGRP1 in mast cells. To examine whether FcεRI-mediated Ras activation was −/− normal, we performed a Ras assay using the GST protein Impaired granule translocation in RasGRP1 BMMCs fused with the Ras-binding domain (RBD) of Raf (GST- Degranulation is thought to be a two-step process. After Raf-RBD). GTP-bound Ras proteins precipitated by GST- granules are translocated to the plasma membrane, they are Raf-RBD were detected by Western blotting with antibodies fused with the plasma membrane to release their contents against H-Ras, N-Ras, and K-Ras, respectively. As shown in (10). To visualize the granule release, we introduced a CD63- Fig. 5 A, the amount of GTP-bound N-Ras precipitated by GFP fusion protein into BMMCs and monitored granule −/− GST-Raf-RBD was reduced in RasGRP1 cells, whereas movement under a confocal microscope as described previ- the amount of GTP-bound K-Ras was similar in WT and ously (10). CD63 is mainly localized in the granules of mast −/− RasGRP1 cells. GTP-bound H-Ras was not detected cells. Before cross-linking with DNP-HSA, mast cell gran- (not depicted). These data indicated that RasGRP1 is likely ules, as revealed by the CD63-GFP fl uorescence, were mostly required for N-Ras activation in FcεRI signaling. in the cytosol. After cross-linking, the majority of granules To determine whether the reduced N-Ras activation was moved close to the plasma membrane in WT BMMCs. In −/− indeed the cause for defective PI3K activation and degranula- contrast, granules in RasGRP1 cells still remained in the tion, we introduced the constitutively activated forms (G12V) cytosol after FcεRI aggregation (Fig. 6 A). After granules are −/− of N-Ras, K-Ras, and H-Ras into RasGRP1 BMMCs fused with the plasma membrane, CD63 can be detected at by retroviral transduction. Stable transductants were selected the cell surface (10). We also examined CD63 surface expres- −/− and expanded. First, we examined whether PI3K activation sion in WT and RasGRP1 cells by staining cells with an could be restored by detecting Akt activation. As shown anti-CD63 antibody followed by FACS analysis. We de- −/− in Fig. 5 B, phosphorylation of Akt in RasGRP1 cells tected an increase of CD63 surface expression on WT cells −/− expressing G12V N-Ras was similar to that in WT cells. but a very minimal increase on RasGRP1 cells (Fig. 6 B). Figure 5. Activation of N-Ras by RasGRP1. (A) Reduced activation of retroviruses expressing constitutively active forms of N-Ras, K-Ras, and −/− −/− N-Ras in RasGRP1 BMMCs. Sensitized WT and RasGRP1 cells were H-Ras were sensitized with anti-DNP IgE and cross-linked with DNP-HSA. activated with DNP-HSA. Lysates were subjected to GST-Raf-RBD precipi- Whole cell lysates were blotted with anti-pAkt, anti-Akt, and anti-HA tation followed by Western blotting with anti–N-Ras and anti–K-Ras antibodies. (C) Effect of a constitutively active Ras on mast cell degranulation. antibodies. (B) Reconstitution of Akt activation by constitutively active Mast cells were transduced as in B. Mast cell degranulation was assayed −/− N-Ras. WT and RasGRP1 BMMCs transduced with empty vector or by measuring the release of β-hexosaminidase. 98 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE Figure 6. Defective granule translocation, microtubule formation, phalloidin (red). (D) Rac1 and RhoA activation. Whole cell lysates were −/− and RhoA activation in RasGRP1 BMMCs. (A) FcεRI-induced gran- subjected to precipitation by GST-PAK-RBD or GST-Rhotekin-RBD beads, −/− ule translocation in WT and RasGRP1 BMMCs. BMMCs expressing respectively. Rac1 and RhoA were detected by Western blotting with an CD63GFP were visualized by confocal microscopy. (B) FcεRI-induced sur- anti-Rac1 or anti-RhoA antibody. Lysates were also blotted with these face CD63 expression analyzed by FACS. (C) FcεRI-induced cytoskeleton antibodies, showing that similar amounts of proteins were used in this rearrangement. Sensitized BMMCs were stimulated with DNP-HSA (+) or assay. One representative of three independent experiments was shown. buffer only (−) and stained with anti–α-tubulin (green) and rhodamine- These data indicated that RasGRP1 is critical for granule pathways (32, 33). We next examined the activation of Rac1 movement in mast cells. Without RasGRP1, mast cells failed and RhoA by using GST-PAK-PBD and GST-Rhotekin to move their granules to the plasma membrane to release RBD fusion proteins to pull down GTP-bound Rac1 their contents. and RhoA. As shown in Fig. 6 D, activation of Rac1 in −/− RasGRP1 cells was similar to that in WT cells. However, −/− −/− Cytoskeleton rearrangement in RasGRP1 BMMCs activation of RhoA was reduced in RasGRP1 cells. These FcεRI engagement induces cytoskeleton rearrangement that results suggested that RasGRP1 plays an important role in includes microtubule formation and disassembly of F-actin FcεRI-mediated RhoA activation. ring. Granule translocation and degranulation require micro- tubule formation (10, 12). To investigate why granules failed DISCUSSION −/− to translocate in RasGRP1 BMMCs, we examined Disruption of RasGRP1 had no eff ect on the proximal sig- formation of microtubule and disassembly of the F-actin naling events after FcεRI engagement. Overall, tyrosine structures by staining BMMCs with anti–α-tubulin and phosphorylation of proteins, phosphorylation of LAT and −/− rhodamine-phalloidin, respectively, before and after FcεRI PLCγ1/γ2, and calcium infl ux were intact in RasGRP1 engagement. Cross-linking of sensitized WT mast cells with mast cells. Calcium infl ux in mast cells is dependent on LAT, DNP-HSA intensifi ed network-like staining (green) of tubu- SLP-76, and PLCγ. LAT or SLP76 defi ciency causes a lin in the cytosol, an indication of the formation of microtu- marked decrease of PLCγ phosphorylation and calcium fl ux bules (Fig. 6 C). In the meantime, F-actin ring, indicated by (4, 34). RasGRP proteins are activated after binding to DAG, phalloidin staining (red), collapsed after stimulation. In acti- a product of PLCγ hydrolysis. Thus, it was not surprising that −/− vated RasGRP1 BMMCs, fl uorescence from anti-tubulin RasGRP1 defi ciency did not aff ect FcεRI-mediated proxi- staining was only seen near the plasma membrane as in un- mal signaling and calcium mobilization. Although PI3K lipid stimulated cells, whereas F-actin disassembly was normal (Fig. products are important for optimal recruitment and activa- 6 C). These results indicated that RasGRP1-mediated signal- tion of PLCγ1 (35), as well as Tec family kinases (36), their ing is required for the formation of microtubules after FcεRI importance in FcεRI-mediated calcium mobilization is not engagement. entirely clear. Mast cells defi cient in Gab2 or Fyn, two criti- Rac and Rho GTPases can regulate cytoskeletal organi- cal molecules in FcεRI-mediated PI3K activation, only have zation in eukaryotic cells (31). Recently, RhoA activation slightly reduced calcium fl ux. It is possible that in mast cells, has been shown to be important for microtubule formation FcεRI-mediated PLCγ activation and calcium mobilization and degranulation in mast cells (10). PI3K and its products are mainly dependent on recruitment of PLCγ to the plasma are required for activation of Rho family proteins in several membrane by LAT. Normal PLCγ phosphorylation and JEM VOL. 204, January 22, 2007 99 −/− calcium mobilization in RasGRP1 mast cells suggested However, the mechanism underlying how PI3K is directly that DAG production is likely normal in these cells. Defec- activated after its localization to membrane has not been tive PI3K activation and mast cell function were less likely clearly characterized in mast cells. Our data suggested that full due to failed DAG production because PMA-induced activa- activation of PI3K after FcεRI engagement requires not only tion of Akt, PKCδ, and PDK1 was still diminished (Fig. 4). membrane recruitment of p85 by Gab2, but also activation RasGRP1 defi ciency aff ects Ras-Erk activation in the by RasGRP1. Our results indicated that RasGRP1 is likely TCR signaling pathway (14), but not in the FcεRI path- involved in PI3K activation through N-Ras. The constitu- way based on our data here. Normal activation of Erk in tively active form of N-Ras could rescue defective Akt ac- −/− RasGRP1 mast cells suggested that Sos or other RasGRP tivation and degranulation. GTP-bound Ras has been shown to interact with the p110 subunit of PI3K and induce its acti- family members might play a major role in FcεRI-mediated Ras-Erk activation. Even though we showed that RasGRP1 vation (8, 39, 40). Effi cient PI3K activation in platelet- was expressed in BMMCs, its abundance was not as high as derived growth factor–induced signaling requires the function in T cells. RasGRP4 is a new member of the RasGRP family of Ras (41). Because RasGRP1 defi ciency did not aff ect and is highly expressed in mast cells (20, 21). C3H/HeJ mice Gab2 phosphorylation and the recruitment of p85, it is likely express a unique isoform of RasGRP4 due to an aberrant that RasGRP1 activates N-Ras, which further induces PI3K splicing. This aberrant splicing produces a dysfunctional pro- activation through its interaction with p110 in FcεRI signal- tein that lacks a DAG-binding domain (C1 domain). These ing (Fig. 7). It is not clear why RasGRP1 is only involved in mice are hyporesponsive to methacholine stimulation via the the activation of N-Ras, not K-Ras or H-Ras. Despite the airway (37), suggesting that RasGRP4 may have an impor- structural similarities among these Ras proteins, their diff er- ential posttranslational modifi cations target them to diff erent tant role in mast cell function, although other genes in these mice can also contribute to the hyporesponsiveness. We microdomains at the membrane or subcellular compartments speculate that RasGRP4 might play a similar role in mast (42, 43). Recent studies suggest that upon T cell activation, cells as RasGRP1 in T cells in Ras-Erk activation. It is also RasGRP1 translocates to the Golgi apparatus to activate Ras possible that FcεRI-mediated Erk activation might be inde- (15). Subsequent studies show that N-Ras is activated upon pendent of RasGRP proteins. In mast cells, Shc is phosphor- engagement of the TCR and PLCγ1 activation, and RasGRP1 ylated upon FcεRI engagement (38). Through binding to may activate N-Ras in the Golgi (44). It remains to be deter- the phosphorylated FcεRI β and γ chains, Shc might recruit mined whether activation of N-Ras by RasGRP1 in mast the Grb2–Sos complex to the plasma membrane to initiate cells occurs in the Golgi or the plasma membrane. Ras-MAPK activation. In contrast with its role in FcεRI-mediated signaling, RasGRP1 is not essential for PI3K and Erk activation in IL-3 Published studies have indicated that PI3K is important in mast cell function (1, 3). Gene inactivation or pharmacologi- or SCF signaling. IL-3 initiates the Jak-Stat pathway, whereas cal inhibition of PI3K impairs FcεRI-mediated degranula- SCF activates the c-Kit receptor tyrosine kinase. Upon phos- tion and cytokine release (25, 26). Recruitment of the p85 phorylation, the IL-3 receptor and c-Kit bind to the Shc– subunit of PI3K by Gab2 is required for its activation (7). Grb2–Sos complex directly to activate the Ras-MAPK pathway Figure 7. A proposed model of RasGRP1 in Fc𝛆 RI-mediated signaling. p110 subunit of PI3K, which binds phosphorylated Gab2, and activates Upon FcεRI cross-linking and PIP hydrolysis, RasGRP1 is recruited to the the PI3K pathway. RasGRP1 also controls RhoA activation indirectly membrane by DAG to activate N-Ras. N-Ras interacts with the catalytic through the PI3K pathway. 100 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE IL-3 for 4 h overnight before stimulation with 20 ng/ml DNP-HSA for the and to p85 to activate the PI3K pathway (45, 46). Why indicated time points in each fi gure. For IL-3 or SCF stimulation, cells RasGRP1 is required for the FcεRI pathway, but not the were starved for 24 h in IMDM medium without IL-3 and stimulated with IL-3– or SCF-mediated pathway, is not clear. It is possible that 20 ng/ml IL-3 or SCF, respectively. IL-3– or SCF-mediated PI3K activation does not require Ras. Mast cell degranulation was performed as described previously (49). For −/− −/− To investigate why RasGRP1 cells failed to degranu- systemic anaphylaxis, WT and RasGRP1 mice were sensitized with anti- late, we analyzed FcεRI-mediated granule translocation and DNP IgE for 24 h and challenged with DNP-HSA for 1.5 min. Blood was collected by cardiac puncture, and histamine concentration in the blood was cytoskeletal rearrangement. Our data indicated that granules determined by ELISA. failed to translocate to the plasma membrane, and the forma- −/− tion of microtubules was defective in RasGRP1 cells after Western blotting. Cells were lysed in RIPA buff er for analysis of whole cell FcεRI engagement. F-actin rearrangement, which is regu- lysates or Brij lysis buff er for immunoprecipitation. For Western blotting, 2+ 2+ lated by Ca and Ca -dependent PKCs (10), was normal in samples were separated by SDS-PAGE and transferred onto nitrocellulose −/− 2+ membranes. After blocking with 1% fi sh gelatin, membranes were incubated RasGRP1 cells. This result was expected because Ca with primary antibodies, washed three times, and probed with either goat fl ux was normal in these cells. Our data showed that RhoA anti–mouse or anti–rabbit Ig conjugated with Alexa Fluor 680 (Invitrogen) or −/− activation was defective in RasGRP1 cells. Vav proteins IRDye 800 (Rockland). Membranes were then visualized and quantifi ed with are GEFs for Rho family GTPases (47) and can be regulated an infrared fl uorescence imaging system (LI-COR Bioscience Odyssey system). by the substrates and products of PI3K (32). Diff erent mem- For most of Western blots, samples were resolved on multiple gels and analyzed bers of the Vav family (Vav1, Vav2, and Vav3) may have dif- by immunoblotting with diff erent antibodies. ferent specifi cities toward diff erent Rho GTPases, such as Numeration of mast cells. Peritoneal fl ushes were stained with toluidine Cdc42, Rac1, and RhoA (48). It is not clear which GEF spe- blue. Percentages of mast cells in the peritoneum were calculated as mast cell −/− cifi cally activates Rac1 and RhoA. In RasGRP1 cells, numbers to total cell numbers in fi ve fi elds under 200× magnifi cation. Rac1 activation was relatively normal, whereas RhoA activa- Skin sections from the back skin and ear dermis were stained with toluidine tion was reduced. It is possible that RasGRP1 activates the blue. Mast cell numbers were counted from at least fi ve sections from −/− each WT (n = 3) and RasGRP1 mouse (n = 3). Data are presented as PI3K pathway, which in turns activates a specifi c GEF mean ± SD. for RhoA, although we cannot exclude the possibility that RasGRP1 is also an exchange factor for RhoA in the FcεRI Calcium infl ux, cytokine production, and PIP measurement. For signaling pathway. calcium infl ux, cells were sensitized with 0.5 μg/ml anti-DNP IgE for 4 h Lyn and Fyn activate two independent pathways upon en- and loaded with 1.5 μM Indo-1 in 1% FBS-HBSS media for 30 min at 30°C. Cells were stimulated with 30 ng/ml DNP-HSA to induce calcium gagement of the FcεRI (6). Activation of the Lyn-Syk-LAT fl ux. The fl uorescence emission ratio at 405–495 nm was monitored by fl ow pathway leads to calcium fl ux and MAPK activation, whereas cytometry. For cytokine production, 2 × 10 anti-DNP IgE sensitized cells the Fyn-Gab2-PI3K pathway is responsible for AKT, PKCδ were stimulated with DNP-HSA for 1 h for RT-PCR and for 8 h for the activation, and degranulation. It has been proposed that there measurement of cytokines released into supernatants using a Bio-Plex cyto- might be cross talks between these two pathways to synergize kine assay (Bio-Rad Laboratories). PIP production was determined using cytokine production and degranulation (6). Our data sug- a protocol described previously (50). gested that RasGRP1 might link LAT phosphorylation and Ras, Rac1, and RhoA activation. For Ras activation, 2 × 10 /ml mast PLCγ activation to PI3K activation, thereby connecting the cells were lysed in a buff er containing 25 mM Hepes, pH 7.5, 150 mM Lyn-initiated pathway to the Fyn-initiated pathway in mast NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mM cells (Fig. 7). In summary, our results indicated that RasGRP1 MgCl , 1 mM EDTA, and 1 mM Na VO . The lysates were incubated with 2 3 4 is an essential regulator of FcεRI-mediated allergic responses. 20 μg GST-Raf-RBD on glutathione beads for 40 min. Beads were washed and boiled in 1× SDS sample buff er. GST-Rhotekin RBD and GST-PAK- MATERIALS AND METHODS PBD fusion protein were provided by K. Burridge (University of North −/− Mice and antibodies. RasGRP1 mice were provided by J.C. Stone Carolina, Chapel Hill, NC). Rac1 and RhoA activation was examined by (University of Alberta, Canada). Mice were housed in specifi c pathogen-free pull-down assays as described previously (51). GTP-bound pan-Ras, H-Ras, conditions, and the use of mice in the experiments described in this study was K-Ras, N-Ras, Rac1, and RhoA were detected by Western blotting with approved by the Duke University Institutional Animal Care Committee. antibodies against each of these proteins, respectively. The following antibodies were used in this study: anti-DNP IgE (SPE-7; Sigma-Aldrich), FcεRIα, and c-Kit (eBioscience); PLCγ2, Erk2, K-Ras, Ras reconstitution by retroviral transduction. Constitutively active forms RhoA, and RasGRP1 (Santa Cruz Biotechnology, Inc.), p85, pTyr (4G10), (G12V) of H-Ras, K-Ras, and N-Ras with an HA tag were subcloned into a retro- PLCγ1, Rac1, and phospho-LATpY191 (Upstate Biotechnology); and pan- viral vector, pMSCV/IRES/Bla. The retroviral plasmids were used to transfect Ras and N-Ras (Calbiochem). AKT, Jnk, p38, PKCδ, PDK1, and other Phoenix cells for retrovirus packaging. 12-d-old BMMCs were transduced with phospho-specifi c antibodies were from Cell Signaling. Anti-LAT (11B12) retroviruses by spin infection. 48 h after transduction, cells were selected with was described previously (49). Anti-RasGRP4 sera were raised by immunizing 5 μg/ml blasticidin and expanded for 10–14 d for subsequent experiments. rabbits with a GST-RasGRP4 fusion protein. Granule translocation and cytoskeletal rearrangement. Examination Mast cell culture, stimulation, and degranulation. Bone marrow cells of granule translocation by confocal microscopy was performed as described −/− were taken from the femurs of WT and RasGRP1 mice and were cul- previously (10). In brief, BMMCs were transduced with pMX-CD63GFP tured in IMDM supplemented with 10% FBS, β-mercaptoethanol, penicillin, retroviruses at day 5 after bone marrow culture in IL-3 medium. At 3 wk and streptomycin in the presence of 5 ng/ml of recombinant IL-3 at 37°C after the initial culture, BMMCs were sensitized with anti-DNP IgE and for 3–6 wk. For stimulation through the FcεRI, 2 × 10 /ml mast cells stimulated with DNP-HSA for 10 min. Cells were immediately fi xed with were sensitized with 0.5 μg/ml anti-DNP IgE in IMDM medium without 4% paraformaldehyde before cytospin and visualized by confocal microscopy. JEM VOL. 204, January 22, 2007 101 For cytoskeletal rearrangement, cells were permeabilized in a buff er con- 16. Dupuy, A.J., K. Morgan, F.C. von Lintig, H. Shen, H. Acar, D.E. taining 0.1% saponin (2% FBS, 1% BSA, and 0.02% sodium azide in PBS) for Hasz, N.A. Jenkins, N.G. Copeland, G.R. Boss, and D.A. Largaespada. 2001. Activation of the Rap1 guanine nucleotide exchange gene, 20 min at room temperature. After cytospin, cells were stained with anti–α- CalDAG-GEF I, in BXH-2 murine myeloid leukemia. J. Biol. Chem. tubulin (Sigma-Aldrich) at 1:50 dilution and incubated with Alexa Fluor 488 276:11804–11811. goat anti–mouse IgG and rhodamine phalloidin (Invitrogen). Confocal mi- 17. Eto, K., R. Murphy, S.W. Kerrigan, A. Bertoni, H. Stuhlmann, T. croscopy was performed using a Zeiss LSM410 confocal system. Nakano, A.D. Leavitt, and S.J. Shattil. 2002. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. −/− We thank Dr. James C. Stone for kindly providing RasGRP1 mice, Ana Sanchez Proc. Natl. Acad. Sci. USA. 99:12819–12824. and Anne Lai for carefully reading the manuscript, and Duke Light Microscopy Core 18. Teixeira, C., S.L. Stang, Y. Zheng, N.S. Beswick, and J.C. Stone. 2003. Facility for confocal microscopy. Integration of DAG signaling systems mediated by PKC-dependent W. Zhang is a scholar of the Leukemia and Lymphoma Society. This work was phosphorylation of RasGRP3. Blood. 102:1414–1420. supported by grants from the National Institutes of Health. 19. Oh-hora, M., S. Johmura, A. Hashimoto, M. Hikida, and T. Kurosaki. The authors have no confl icting fi nancial interests. 2003. Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-γ2 to Ras in B cell receptor signaling. J. Exp. Submitted: 31 July 2006 Med. 198:1841–1851. Accepted: 1 December 2006 20. Reuther, G.W., Q.T. Lambert, J.F. Rebhun, M.A. Caligiuri, L.A. Quilliam, and C.J. Der. 2002. RasGRP4 is a novel Ras activator R E F E R E N C E S isolated from acute myeloid leukemia. J. Biol. Chem. 277:30508–30514. 1. Kinet, J.P. 1999. The high-affi nity IgE receptor (FcεRI): from physiology 21. Yang, Y., L. Li, G.W. Wong, S.A. Krilis, M.S. Madhusudhan, A. Sali, to pathology. Annu. Rev. Immunol. 17:931–972. and R.L. Stevens. 2002. RasGRP4, a new mast cell-restricted Ras 2. Ravetch, J.V. 1994. Fc receptors: rubor redux. Cell. 78:553–560. guanine nucleotide-releasing protein with calcium- and diacylglycerol- 3. Rivera, J., J.R. Cordero, Y. Furumoto, C. Luciano-Montalvo, C. binding motifs. J. Biol. Chem. 277:25756–25774. Gonzalez-Espinosa, M. Kovarova, S. Odom, and V. Parravicini. 2002. 22. Galli, S.J. 2000. Mast cells and basophils. Curr. Opin. Hematol. Macromolecular protein signaling complexes and mast cell responses: 7:32–39. a view of the organization of IgE-dependent mast cell signaling. Mol. 23. Boudreau, R.T., D.W. Hoskin, and T.J. Lin. 2004. Phosphatase in- Immunol. 38:1253–1258. hibition potentiates IL-6 production by mast cells in response to 4. Saitoh, S., R. Arudchandran, T.S. Manetz, W. Zhang, C.L. Sommers, FcεRI-mediated activation: involvement of p38 MAPK. J. Leukoc. Biol. P.E. Love, J. Rivera, and L.E. Samelson. 2000. LAT is essential for 76:1075–1081. FcεRI-mediated mast cell activation. Immunity. 12:525–535. 24. Ishizuka, T., N. Terada, P. Gerwins, E. Hamelmann, A. Oshiba, G.R. 5. Samelson, L.E. 2002. Signal transduction mediated by the T cell an- Fanger, G.L. Johnson, and E.W. Gelfand. 1997. Mast cell tumor necrosis tigen receptor: the role of adapter proteins. Annu. Rev. Immunol. factor alpha production is regulated by MEK kinases. Proc. Natl. Acad. 20:371–394. Sci. USA. 94:6358–6363. 6. Parravicini, V., M. Gadina, M. Kovarova, S. Odom, C. Gonzalez- 25. Ali, K., A. Bilancio, M. Thomas, W. Pearce, A.M. Gilfi llan, C. Tkaczyk, Espinosa, Y. Furumoto, S. Saitoh, L.E. Samelson, J.J. O’Shea, and J. N. Kuehn, A. Gray, J. Giddings, E. Peskett, et al. 2004. Essential role Rivera. 2002. Fyn kinase initiates complementary signals required for for the p110δ phosphoinositide 3-kinase in the allergic response. Nature. IgE-dependent mast cell degranulation. Nat. Immunol. 3:741–748. 431:1007–1011. 7. Gu, H., K. Saito, L.D. Klaman, J. Shen, T. Fleming, Y. Wang, J.C. 26. Barker, S.A., K.K. Caldwell, A. Hall, A.M. Martinez, J.R. Pfeiff er, Pratt, G. Lin, B. Lim, J.P. Kinet, and B.G. Neel. 2001. Essential role for J.M. Oliver, and B.S. Wilson. 1995. Wortmannin blocks lipid and pro- Gab2 in the allergic response. Nature. 412:186–190. tein kinase activities associated with PI 3-kinase and inhibits a subset 8. Rodriguez-Viciana, P., P.H. Warne, R. Dhand, B. Vanhaesebroeck, of responses induced by Fc epsilon R1 cross-linking. Mol. Biol. Cell. I. Gout, M.J. Fry, M.D. Waterfi eld, and J. Downward. 1994. 6:1145–1158. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 27. Huber, M., C.D. Helgason, J.E. Damen, L. Liu, R.K. Humphries, and 370:527–532. G. Krystal. 1998. The src homology 2-containing inositol phosphatase 9. Wick, M.J., F.J. Ramos, H. Chen, M.J. Quon, L.Q. Dong, and F. (SHIP) is the gatekeeper of mast cell degranulation. Proc. Natl. Acad. Sci. Liu. 2003. Mouse 3-phosphoinositide-dependent protein kinase-1 un- USA. 95:11330–11335. dergoes dimerization and trans-phosphorylation in the activation loop. 28. Vanhaesebroeck, B., and D.R. Alessi. 2000. The PI3K-PDK1 connection: J. Biol. Chem. 278:42913–42919. more than just a road to PKB. Biochem. J. 346:561–576. 10. Nishida, K., S. Yamasaki, Y. Ito, K. Kabu, K. Hattori, T. Tezuka, 29. Kitaura, J., K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, H. Nishizumi, D. Kitamura, R. Goitsuka, R.S. Geha, et al. 2005. and T. Kawakami. 2000. Akt-dependent cytokine production in mast FcεRI-mediated mast cell degranulation requires calcium-independent cells. J. Exp. Med. 192:729–740. microtubule-dependent translocation of granules to the plasma membrane. 30. Le Good, J.A., W.H. Ziegler, D.B. Parekh, D.R. Alessi, P. Cohen, J. Cell Biol. 170:115–126. and P.J. Parker. 1998. Protein kinase C isotypes controlled by phos- 11. Martin-Verdeaux, S., I. Pombo, B. Iannascoli, M. Roa, N. Varin-Blank, phoinositide 3-kinase through the protein kinase PDK1. Science. J. Rivera, and U. Blank. 2003. Evidence of a role for Munc18-2 and 281:2042–2045. microtubules in mast cell granule exocytosis. J. Cell Sci. 116:325–334. 31. Etienne-Manneville, S., and A. Hall. 2002. Rho GTPases in cell 12. Smith, A.J., J.R. Pfeiff er, J. Zhang, A.M. Martinez, G.M. Griffi ths, biology. Nature. 420:629–635. and B.S. Wilson. 2003. Microtubule-dependent transport of secretory 32. Han, J., K. Luby-Phelps, B. Das, X. Shu, Y. Xia, R.D. Mosteller, vesicles in RBL-2H3 cells. Traffi c. 4:302–312. U.M. Krishna, J.R. Falck, M.A. White, and D. Broek. 1998. Role of 13. Ebinu, J.O., D.A. Bottorff , E.Y. Chan, S.L. Stang, R.J. Dunn, and J.C. substrates and products of PI 3-kinase in regulating activation of Rac- Stone. 1998. RasGRP, a Ras guanyl nucleotide-releasing protein with related guanosine triphosphatases by Vav. Science. 279:558–560. calcium- and diacylglycerol-binding motifs. Science. 280:1082–1086. 33. Saci, A., and C.L. Carpenter. 2005. RhoA GTPase regulates B cell re- 14. Dower, N.A., S.L. Stang, D.A. Bottorff , J.O. Ebinu, P. Dickie, H.L. ceptor signaling. Mol. Cell. 17:205–214. Ostergaard, and J.C. Stone. 2000. RasGRP is essential for mouse thy- 34. Pivniouk, V.I., T.R. Martin, J.M. Lu-Kuo, H.R. Katz, H.C. Oettgen, mocyte diff erentiation and TCR signaling. Nat. Immunol. 1:317–321. and R.S. Geha. 1999. SLP-76 defi ciency impairs signaling via the high- 15. Bivona, T.G., I. Perez de Castro, I.M. Ahearn, T.M. Grana, V.K. Chiu, affi nity IgE receptor in mast cells. J. Clin. Invest. 103:1737–1743. P.J. Lockyer, P.J. Cullen, A. Pellicer, A.D. Cox, and M.R. Philips. 35. Bae, Y.S., L.G. Cantley, C.S. Chen, S.R. Kim, K.S. Kwon, and S.G. 2003. Phospholipase Cγ activates Ras on the Golgi apparatus by means Rhee. 1998. Activation of phospholipase C-γ by phosphatidylinositol of RasGRP1. Nature. 424:694–698. 3,4,5-trisphosphate. J. Biol. Chem. 273:4465–4469. 102 RASGRP1 IN FCεRI SIGNALING AND MAST CELL FUNCTION | Liu et al. ARTICLE 36. Scharenberg, A.M., O. El-Hillal, D.A. Fruman, L.O. Beitz, Z. Li, 43. Mor, A., and M.R. Philips. 2006. Compartmentalized ras/mapk signaling. S. Lin, I. Gout, L.C. Cantley, D.J. Rawlings, and J.P. Kinet. 1998. Annu. Rev. Immunol. 24:771–800. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase- 44. Perez de Castro, I., T.G. Bivona, M.R. Philips, and A. Pellicer. 2004. dependent calcium signaling pathway: a target for SHIP-mediated in- Ras activation in Jurkat T cells following low-grade stimulation of hibitory signals. EMBO J. 17:1961–1972. the T-cell receptor is specifi c to N-Ras and occurs only on the Golgi 37. Li, L., Y. Yang, G.W. Wong, and R.L. Stevens. 2003. Mast cells in apparatus. Mol. Cell. Biol. 24:3485–3496. airway hyporesponsive C3H/HeJ mice express a unique isoform of the 45. Roskoski, R., Jr. 2005. Signaling by Kit protein-tyrosine kinase–the signaling protein Ras guanine nucleotide releasing protein 4 that is unre- stem cell factor receptor. Biochem. Biophys. Res. Commun. 337:1–13. sponsive to diacylglycerol and phorbol esters. J. Immunol. 171:390–397. 46. Martinez-Moczygemba, M., and D.P. Huston. 2003. Biology of com- 38. Jabril-Cuenod, B., C. Zhang, A.M. Scharenberg, R. Paolini, R. mon β receptor-signaling cytokines: IL-3, IL-5, and GM-CSF. J. Allergy Numerof, M.A. Beaven, and J.-P. Kinet. 1996. Syk-dependent phos- Clin. Immunol. 112:653–665. phorylation of Shc. A potential link between FcεRI and the Ras/ 47. Crespo, P., K.E. Schuebel, A.A. Ostrom, J.S. Gutkind, and X.R. Bustelo. mitogen-activated protein kinase signaling pathway through SOS and 1997. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP ex- Grb2. J. Biol. Chem. 271:16268–16272. change by the vav proto-oncogene product. Nature. 385:169–172. 39. Marte, B.M., P. Rodriguez-Viciana, S. Wennstrom, P.H. Warne, and J. 48. Abe, K., K.L. Rossman, B. Liu, K.D. Ritola, D. Chiang, S.L. Campbell, Downward. 1997. R-Ras can activate the phosphoinositide 3-kinase but K. Burridge, and C.J. Der. 2000. Vav2 is an activator of Cdc42, Rac1, not the MAP kinase arm of the Ras eff ector pathways. Curr. Biol. 7:63–70. and RhoA. J. Biol. Chem. 275:10141–10149. 40. Kodaki, T., R. Woscholski, B. Hallberg, P. Rodriguez-Viciana, J. 49. Zhu, M., Y. Liu, S. Koonpaew, O. Granillo, and W. Zhang. 2004. Downward, and P.J. Parker. 1994. The activation of phosphatidylinositol Positive and negative regulation of FcεRI-mediated signaling by the 3-kinase by Ras. Curr. Biol. 4:798–806. adaptor protein LAB/NTAL. J. Exp. Med. 200:991–1000. 41. Klinghoff er, R.A., B. Duckworth, M. Valius, L. Cantley, and A. 50. Manetz, T.S., C. Gonzalez-Espinosa, R. Arudchandran, S. Xirasagar, Kazlauskas. 1996. Platelet-derived growth factor-dependent activa- V. Tybulewicz, and J. Rivera. 2001. Vav1 regulates phospholipase tion of phosphatidylinositol 3-kinase is regulated by receptor binding Cγ activation and calcium responses in mast cells. Mol. Cell. Biol. of SH2-domain-containing proteins which infl uence Ras activity. Mol. 21:3763–3774. Cell. Biol. 16:5905–5914. 51. Liu, B.P., and K. Burridge. 2000. Vav2 activates Rac1, Cdc42, and 42. Prior, I., and J. Hancock. 2001. Compartmentalization of Ras proteins. RhoA downstream from growth factor receptors but not β1 integrins. J. Cell Sci. 114:1603–1608. Mol. Cell. Biol. 20:7160–7169. JEM VOL. 204, January 22, 2007 103

Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Jan 22, 2007

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