Rap1 Is Activated by Erythropoietin or Interleukin-3 and Is Involved in Regulation of beta 1 Integrin-mediated Hematopoietic Cell Adhesion*

Ayako AraiDagger , Yurika NosakaDagger §, Eiichiro KandaDagger , Koh YamamotoDagger , Nobuyuki Miyasaka§, and Osamu MiuraDagger

From the Dagger  Department of Hematology and Oncology and the § Department of Bioregulatory Medicine and Rheumatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113, Japan

Received for publication, May 29, 2000, and in revised form, December 5, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta 1 integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta 1 integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas U73122, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta 1 integrin-mediated hematopoietic cell adhesion.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The growth and differentiation of hematopoietic progenitor cells are regulated by hematopoietic cytokines, including erythropoietin (Epo)1 and interleukin (IL)-3, as well as through direct interaction with the bone marrow microenvironment, composed of stromal cells and extracellular matrix components, such as fibronectin. The hematopoietic cytokine receptors mainly couple with Jak2, a member of the JAK family of tyrosine kinases, to activate various signal transduction pathways, including those involving the Ras/Erk activation cascade, Stats, phosphatidylinositol 3'-kinase, and phospholipase C (PLC)-gamma (1). Most of the hematopoietic cytokines, such as stem cell factor, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), thrombopoietin, and Epo, also activate adhesion of hematopoietic cells to extracellular matrix components through integrins of the beta 1 subfamily, mainly VLA-4 (alpha 4beta 1) and VLA-5 (alpha 5beta 1) (2-5). However, the mechanisms by which receptors for these cytokines transduce signals to activate integrins (inside-out signaling) have remained elusive.

CrkL is a member of the Crk family of adaptor proteins originally identified as homologues of the product of the v-crk oncogene and is most abundantly expressed in hematopoietic cells (6). CrkL has the domain structure Src homology (SH)2-SH3-SH3 and has been shown to bind through its N-terminal SH3 domain with various signaling molecules including Sos1 and C3G, two guanine nucleotide exchange factors (GEFs) for the Ras family of small GTP-binding proteins (7, 8). We have demonstrated previously that CrkL is involved in hematopoietic cytokine receptor signaling because CrkL becomes tyrosine-phosphorylated in hematopoietic cells in response to stimulation with Epo or IL-3 and forms complexes with several tyrosine-phosphorylated signaling molecules, such as Cbl, Shc, and SHP-2 (9). Furthermore, we showed that CrkL is involved through its interaction with C3G in activation of the Ras/Erk signaling pathway leading to the induction of c-fos gene expression in response to Epo or IL-3 (10). Intriguingly, hematopoietic cells overexpressing CrkL or C3G showed an enhanced adhesion through beta 1 integrins VLA-4 and VLA-5 in response to cytokines, whereas cells expressing a dominant negative mutant of CrkL or C3G showed an impairment in adhesion (11). The CrkL-mediated enhancement of hematopoietic cell adhesion, also reported by others (12, 13), was observed without changes in expression levels of these integrins, thus suggesting that it is through the increase in ligand binding activity of integrins (11). These observations, thus, indicate that CrkL as well as C3G is involved in cytokine signaling leading to the activation of hematopoietic cell adhesion through beta 1 integrins. However, the CrkL-mediated activation of hematopoietic cell adhesion was independent of Ras activation (11). Thus, it has remained unknown how the CrkL-C3G complex activates the downstream signaling pathways leading to integrin activation.

Rap1 is a small GTPase of the Ras family which was first identified as a homologue of Ras (14) and was also identified independently by its ability to induce a flat revertant phenotype in K-Ras-transformed cells (15). Two highly homologous forms of Rap1, Rap1A and Rap1B, have been identified (16), but their functional difference has been unclear. Recent studies have shown that Rap1 is activated in fibroblasts after stimulation with a variety of growth factors, including platelet-derived growth factor, epidermal growth factor, and lysophosphatidic acid (17, 18). Rap1 is also activated after stimulation of the T cell or B cell antigen receptor in lymphocytes (19, 20) and by stimulation with thrombin in platelets (21). Depending on cell types, different types of intracellular second messengers, including calcium, diacylglycerol, and cyclic AMP, have been implicated in Rap1 activation (17, 18). Recently identified, two GEFs for Rap1, Epac (22), which is directly activated by cyclic AMP, and CalDAG-GEFI (23), which is sensitive to both calcium and diacylglycerol, have been supposed to mediate Rap1 activation in response to these second messengers. C3G, which forms a complex with CrkL, also efficiently activates Rap1 in vitro as well as when expressed in COS cells (24). Furthermore, C3G as well as CrkL has recently been implicated in Rap1 activation induced by stimulation with nerve growth factor (25), interferon-gamma (26), and hepatocyte growth factor (27) or by stimulation of the T cell antigen receptor (19). With an effector domain that is nearly identical to Ras and binds to many of the same effectors, Rap1 has been hypothesized to have antagonistic effects on the downstream signaling events of Ras. However, roles for Rap1 in physiological signaling pathways largely remain elusive (17, 18).

Previously, M'Rabet et al. (28) reported that GM-CSF, a hematopoietic cytokine that shares a receptor chain with IL-3, stimulates Rap1 in mature neutrophils. Rap1 is also activated in neutrophils by other stimuli that are involved in neutrophil activation, including fMet-Leu-Phe, platelet-activating factor, and IgG-coated particles (28). However, the signaling mechanisms for GM-CSF-induced Rap1 activation in neutrophils and its physiological significance remain to be understood. Furthermore, although Rap1 has been shown to be activated in highly differentiated and specialized hematopoietic cells, such as mature neutrophils (28) and platelets (21), it has remained to be determined whether Rap1 is involved in cytokine receptor signaling in hematopoietic progenitor cells.

In the present study, we have examined the possible involvement of Rap1 in cytokine receptor signaling and reveal that Epo as well as IL-3 activates Rap1 in hematopoietic 32D cells, most likely through the signaling pathways involving the CrkL-C3G complex and PLC-gamma . Furthermore, the present study indicates that Rap1 is involved in the regulation of hematopoietic cell adhesion through beta 1 integrins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- A clone of IL-3-dependent 32D cells expressing the wild-type murine Epo receptor (EpoR), 32D/EpoR-Wt, was described previously (29) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 1 unit/ml human recombinant Epo. 32DE/TA cells, which inducibly express the tetracycline transactivator (tTA) when withdrawn from tetracycline, and 32DE/Tet-CrkL or 32DE/Tet-C3G cells, which inducibly overexpress CrkL or C3G, respectively, when withdrawn from tetracycline, were also described previously (11). A human leukemic cell line expressing the endogenous EpoR, UT-7 (30), was kindly provided by Dr. Norio Komatsu (Jichi Medical School, Tochigi, Japan). COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

Recombinant human Epo was kindly provided by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). Recombinant murine IL-3 was purchased from PeproTech Inc. (Rocky Hill, NJ). Phorbol 12-myristate 13-acetate (PMA), ionomycin, and forskolin were purchased from Sigma Chemicals (St. Louis, MO). U73122 and U73343 were from Calbiochem (La Jolla, CA). Antibodies against Rap1A, CrkL, C3G, and EpoR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphotyrosine (4G10), Erk1/2 (Erk1/2-CT), and PLC-gamma 1 (05-163) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-Erk (Thr-202/Tyr-204) and anti-phospho-Stat5 (Tyr-694) were purchased from New England Biolabs (Beverly, MA). A monoclonal antibody against the T7 epitope was purchased from Novagen (Madison, WI). Monoclonal antibodies against murine beta 1 integrin (HA2/5, hamster IgM, kappa ) and murine alpha 5 integrin (5H10-27, Rat IgG2a, kappa ) were purchased from Pharmingen (San Diego, CA). A monoclonal antibody against murine alpha 4 integrin (428, Rat IgG2a, kappa ) and control murine monoclonal antibodies (IgM, kappa , and IgG2a, kappa ) were purchased from Seikagaku Corp. (Tokyo, Japan) and Sigma Chemicals, respectively.

Plasmids-- Expression plasmids for human CrkL, pSG-CrkL (31), and dominant negative R-Ras, pcDNA-R-Ras-43N (32), were kindly provided by Dr. John Groffen (Childrens Hospital Los Angeles, Los Angeles, CA) and Dr. Erkki Ruoslahti (La Jolla Cancer Research Center, La Jolla, CA), respectively. An expression plasmid for C3G (pcDNA-C3G) as well as those for its activated and dominant negative mutants, pcDNA-C3G-dN and pcDNA-C3G-dSS, respectively, was described previously (11). Expression plasmids for dominant negative and constitutively activated mutants of Rap1/K-Rev1 (33) tagged with the T7 epitope, pSRalpha -T7-Rap1A-17N and pSRalpha -T7-Rap1A-63E, respectively, were kindly provided by Drs. Makoto Noda and Masakazu Hattori (Kyoto University, Kyoto, Japan). For construction of pTet-Rap1A-63E, a tetracycline-responsive expression plasmid for Rap1A-63E, the BamHI/EcoRV fragment of pSRalpha -T7-Rap1A-63E coding for T7-tagged Rap1A-63E, was excised and subcloned between the BamHI and HindIII sites in pcDNA3 (Invitrogen, San Diego, CA) to give pcDNA-Rap1A-63E. The region coding for T7-tagged Rap1A-63E was then excised from pcDNA3-Rap1A-63E by digestion with both HindIII and XbaI and subcloned between the HindIII and SpeI sites of pTet-Splice to give pTet-Rap1A-63E. The pGEX-RalGDS-RBD plasmid encoding a glutathione S-transferase (GST) fusion protein containing the 97-amino acid Rap1 binding domain (RBD) of the RalGDS protein (34) was kindly provided by Dr. Johannes L. Bos (Utrecht University, Utrecht, The Netherlands). A control luciferase plasmid, pGL-cont, was purchased from Promega (Madison, WI).

Transfection-- Transfection for stable expression was carried out essentially as described previously (29). In brief, 32DE/TA cells were transfected with 10 µg of pTet-Rap1A-63E along with 1 µg of pMAM2-BSD, purchased from Funakoshi (Tokyo, Japan), by electroporation at 960 microfarads and 300 V, followed by selection in medium containing blasticidin-S (Funakoshi) and 500 ng/ml tetracycline. Six clones transfected with pTet-Rap1A-63E were isolated by limiting dilution and examined for the induction of Rap1A-63E expression by anti-T7 immunoblotting of cell lysates prepared after withdrawal from tetracycline for 24 h. The clone inducibly expressing the highest level of Rap1A-63E, 32DE/TA-Rap1A-63E, was selected for the subsequent studies.

Similarly, a clone of 32D/EpoR-Wt, 32DE/CrkL, which constitutively overexpresses CrkL, was obtained by transfection of pSG-CrkL.

Transfection of expression plasmids into COS-7 cells was carried out using the LipofectAMINE reagent (Life Technologies, Inc.), as described previously (35). 2 days after transfection, cells were harvested for analysis.

Immunoprecipitation and Rap1 Activation Assay-- For immunoprecipitation experiments, cells were lysed in a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each aprotinin and leupeptin. Cell lysates were subjected to immunoprecipitation as described previously (36).

Rap1 activation in cells was determined by using an activation-specific probe, essentially as described by Zwartkruis et al. (37). In brief, cells were lysed in a lysis buffer containing 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2.5 mM MgCl2, 10 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each aprotinin and leupeptin. Cell lysates were incubated with the GST-RalGDS-RBD fusion protein, prepared as described previously and precoupled to glutathione-agarose beads. After incubation for 45 min at 4 °C, beads were washed three times in lysis buffer. Rap1-GTP bound to beads was eluted by heating at 100 °C for 5 min in 1 × Laemmli's sample buffer and detected by immunoblotting with anti-Rap1 antibody using the enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Buckinghamshire, U. K.). For immunoblot analysis of total cell lysates, samples were prepared by mixing an aliquot of cell lysates with an equal volume of 2 × Laemmli's sample buffer and heating at 100 °C for 5 min. All Rap1 activation assays were performed at least three times with reproducible results. Quantification of blots was performed with NIH Image software.

Cell Adhesion Assays-- Adhesion assays of transiently transfected cells were performed essentially as described previously (11). In brief, 32D/EpoR-Wt and UT-7 cells were electroporated with the indicated amounts of relevant plasmids and 1 µg of a control luciferase plasmid, pGL-cont. The total amount of expression plasmids for each transfection was adjusted to become equal by adding an empty plasmid. After a recovery period of 1 day in medium containing 1 unit/ml Epo, cells were plated in triplicate on wells coated with 5 µg/ml fibronectin and incubated at 37 °C for 30 min. After wells were washed three times to remove unbound cells, cells remaining attached to the wells were measured by the luciferase assay. After subtraction of background cell binding to bovine serum albumin-coated wells, the percentage of adherent cells was determined by dividing the luciferase activity of the adherent cells by that of the initial cell input. For adhesion assays of stably transfected clones, cells were labeled with 5 µM 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy fluorescein acetoxymethyl ester (Dojindo, Kumamoto, Japan) at 37 °C for 1 h. After being washed three times, fluorescently labeled cells were subjected to cell adhesion assay as described above. Cells were measured by Cytofluor II fluorescent plate reader (PerSeptive Biosystems, Foster City, CA). Alternatively, unlabeled cells were subjected to cell adhesion assays and measured by using the sodium XTT colorimetric assay kit (Boehringer Mannheim, Indianapolis, IN), as described previously (11). All cell adhesion assays under "Results" were carried out using fibronectin-coated plates unless described otherwise and repeated at least three times, and the results were reproducible.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IL-3 or Epo Stimulation Induces Rap1 Activation in Hematopoietic Cells-- Previously, we have shown that CrkL is involved in hematopoietic cytokine receptor signaling and plays a role in activation of integrin-mediated hematopoietic cell adhesion through its interaction with C3G (9, 11), a GEF that most efficiently activates Rap1 (24). To address the possibility that Rap1 may be involved in the CrkL-mediated cytokine receptor signaling pathway regulating hematopoietic cell adhesion, we first examined whether IL-3 or Epo induces Rap1 activation in hematopoietic cells. As shown in Fig. 1A, the affinity purification assay using the GST-RalGDS-RBD, as described under "Experimental Procedures," demonstrated that IL-3 as well as Epo stimulation induces activation of Rap1 in 32D/EpoR-Wt cells, a clone of murine IL-3-dependent 32D cells expressing the EpoR (29). The Rap1 activation induced by IL-3 or Epo was observed as early as 1 min after stimulation, increased to plateau at 5-20 min, and was sustained as long as 60 min. As shown in Fig. 1B, Rap1 was significantly activated when cells were stimulated with Epo or IL-3 at a concentration as low as 1 unit/ml or 1 ng/ml, respectively. When stimulated with a higher concentration of Epo (10 units/ml) or IL-3 (10 ng/ml), a further increase in Rap1 activity was observed. Anti-Rap1 blotting of total cell lysates showed that the total amount of Rap1 in the cells did not change significantly after stimulation with IL-3 or Epo (Fig. 1, lower panels). These results thus demonstrate that IL-3 and Epo induce time- and dose-dependent activation of Rap1 in hematopoietic cells.


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Fig. 1.   Epo as well as IL-3 activates Rap1 in 32D/EpoR-Wt cells. 32D/EpoR-Wt cells, a clone of the IL-3-dependent 32D cell line expressing the EpoR, were starved from Epo overnight in serum-free medium and stimulated with 10 units/ml Epo or 1 ng/ml IL-3 for various times as indicated in A or stimulated with the indicated concentrations of Epo (units/ml) or IL-3 (ng/ml) for 15 min in B. Activated Rap1 (Rap1-GTP) was affinity purified from cell lysates using a GST-RalGDS-RBD fusion protein, as described under "Experimental Procedures." Eluates from the precipitates (upper panels) or total cell lysates (lower panels) were subsequently subjected to immunoblotting with anti-Rap1 antibody. Relative Rap1 activities, determined by densitometric analysis, are shown between the two blots.

Involvement of CrkL and C3G in IL-3- or Epo-induced Activation of Rap1-- To examine whether CrkL is involved in cytokine-induced activation of Rap1 in hematopoietic cells, we next examined the cytokine-induced activation of Rap1 in a clone of 32D/EpoR-Wt cells, 32DE/Tet-CrkL, which inducibly overexpresses CrkL when withdrawn from tetracycline (11). As shown in Fig. 2A and B, the Rap1 activation induced by IL-3 as well as the basal level of Rap1 activity in 32DE/Tet-CrkL was augmented when cells were depleted overnight from tetracycline. Anti-Rap1 and anti-CrkL blotting of total cell lysates confirmed that the depletion of tetracycline increased the expression level of CrkL but not that of Rap1 in 32DE/Tet-CrkL cells. Similarly, Epo-induced activation of Rap1 was augmented when 32DE/Tet-CrkL cells were induced to overexpress CrkL (data not shown). These results strongly suggest that CrkL is involved, most likely through its interaction with C3G, in IL-3- and Epo-induced activation of Rap1.


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Fig. 2.   Cytokine-induced activation of Rap1 is augmented in cells inducibly overexpressing CrkL or C3G. A and B, a clone of 32D/EpoR-Wt cells, 32DE/Tet-CrkL, which overexpresses CrkL when withdrawn from tetracycline, was starved from Epo overnight in serum-free medium containing 100 ng/ml tetracycline (Tet+) or without tetracycline (Tet-), as indicated. Cells were then stimulated with 1 ng/ml IL-3 for the indicated times in A or with the indicated concentrations (ng/ml) of IL-3 for 15 min in B and subjected to the Rap1 activation assays. Eluates from the GST-RalGDS-RBD precipitates (top panels) or total cell lysates (middle and bottom panels) were subsequently subjected to immunoblotting with anti-Rap1 or anti-CrkL antibody, as indicated. Relative Rap1 activities are shown between the two anti-Rap1 blots. C, 32DE/Tet-C3G cells, which overexpress C3G when withdrawn from tetracycline, were starved from Epo overnight in serum-free medium containing 100 ng/ml tetracycline (Tet+) or without tetracycline (Tet-), as indicated. Cells were then stimulated with 10 units/ml Epo (E) or 1 ng/ml IL-3 (I), as indicated, for 15 min and subjected to the Rap1 activation assays. Eluates from the GST-RalGDS-RBD precipitates (top panel) or total cell lysates (middle and bottom panels) were subsequently subjected to immunoblotting with anti-Rap1, anti-phospho-Erk, or anti-C3G antibody, as indicated.

To confirm that C3G is also involved in cytokine-induced Rap1 activation, we examined 32DE/Tet-C3G cells, which overexpress C3G when withdrawn from tetracycline (11). As shown in Fig. 2C, the Rap1 activation induced by Epo or IL-3 was augmented significantly when C3G was overexpressed. Anti-phospho-Erk blotting of total cell lysates demonstrated that the Epo- or IL-3-induced activation of Erk is also enhanced when C3G was overexpressed, which is in agreement with our previous observation that C3G is also involved in cytokine-induced activation of the Ras/Erk signaling pathway. Together, these results strongly suggest that the CrkL-C3G complex is involved in Epo- and IL-3-induced activation of Rap1.

Rap1 Is Involved in CrkL-induced Activation of Integrin-mediated Adhesion of Hematopoietic Cells-- To explore the possibility that CrkL-mediated activation of hematopoietic cell adhesion is mediated by Rap1, we first employed Rap1A-17N, a putative dominant negative mutant of Rap1 (25, 38). However, because the dominant negative effect of Rap1A-17N has been disputed by recent in vitro studies (39), we first examined whether Rap1A-17N inhibits Rap1 activation mediated by C3G in COS cells. As shown in Fig. 3A, when transiently overexpressed in COS cells, the activated form of wild-type Rap1, but not that of Rap1A-17N, was detected by affinity purification assays with GST-RalGDS-RBD. In accordance with a previously report (24), when C3G was coexpressed, the activated form of wild-type Rap1 was increased drastically. However, the C3G-induced activation of wild-type Rap1 was abrogated by coexpression of Rap1A-17N (Fig. 3A, upper panel), which indicates that Rap1A-17N inhibits the activation of Rap1 by C3G in a dominant negative manner. Anti-T7 blotting of total cell lysates demonstrated that the expression level of wild-type Rap1 is significantly higher than that of Rap1A-17N in repeated experiments (Fig. 3A, middle panel, and data not shown). In addition, when coexpressed with and activated by C3G, the expression level of Rap1 was found to be significantly increased in repeated experiments (Fig. 3A, middle panel, and data not shown). It is thus possible that the activated form of Rap1 may be more stable than the inactive form, although further studies are required to address this possibility. Irrespective of this, these results have demonstrated unequivocally that Rap1A-17N inhibits C3G-mediated Rap1 activation in cells.


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Fig. 3.   Involvement of Rap1 in CrkL-enhanced hematopoietic cell adhesion through beta 1 integrins to fibronectin. A, COS-7 cells were transfected with 0.5 µg of pcDNA-C3G (C3G), pSRalpha -T7-Rap1A-17N (Rap1-17N), or pSRalpha -T7-Rap1A-Wt (Rap1-Wt), as indicated. The total amount of expression plasmids for each transfection was adjusted to become equal by the addition of pcDNA3. 2 days after transfection, cells were lysed and subjected to an affinity purification assay with GST-RalGDS-RBD. Eluates (top panel) and total cell lysates (middle and lower panels) were then subjected to anti-T7 or anti-C3G immunoblotting, as indicated. B, 32D/EpoR-Wt cells were transfected with 10 µg of pSG-CrkL (CrkL), 20 µg of pSRalpha -T7-Rap1A-17N (Rap1-17N), or 20 µg of pcDNA-R-Ras-43N (R-Ras-43N), as indicated, along with 1 µg of pGL-cont. Transiently transfected cells were subjected to the cell adhesion assay measured by the luciferase assay as described under "Experimental Procedures." C, 32D/EpoR-Wt cells were transfected with the indicated amount of pSRalpha -T7-Rap1A-63E along with 1 µg of pGL-cont. Transiently transfected cells were subjected to the cell adhesion assay as measured by the luciferase assay. D, 32DE/TA-Rap1A-63E cells were cultured overnight with the indicated concentrations of tetracycline, fluorescently labeled, and subjected to the cell adhesion assays as measured by fluorometry as described under "Experimental Procedures." Anti-T7 immunoblotting of the GST-RalGDS-RBD precipitates obtained from lysates of cells cultured under the same conditions is also shown. E, 32DE/TA-Rap1A-63E cells, cultured overnight with or without tetracycline as indicated, were allowed to attach to wells coated with the indicated concentrations of fibronectin for the cell adhesion assays as measured by the XTT colorimetric method. Anti-T7 immunoblotting of the GST-RalGDS-RBD precipitates obtained from lysates of cells cultured under the same conditions is also shown. F, 32DE/TA-Rap1A-63E cells, cultured overnight with or without tetracycline (Tet.) as indicated, were fluorescently labeled, preincubated for 15 min at room temperature with anti-beta 1 integrin monoclonal antibody (Anti-beta 1) or mouse IgMkappa (IgM) as a control as indicated, and subjected to the cell adhesion assays as measured by fluorometry. G, 32DE/TA-Rap1A-63E cells, cultured overnight without tetracycline, were fluorescently labeled, preincubated for 15 min at room temperature with anti-alpha 4, anti-alpha 5, or anti-beta 1 integrin monoclonal antibodies, as indicated, or with mouse IgG2akappa (IgG) or IgMkappa (IgM) as a control as indicated, and subjected to the cell adhesion assays as measured by fluorometry.

Having established the dominant negative effect of Rap1A-17N in cells, we examined the effect of this mutant on CrkL-enhanced cell adhesion to fibronectin. As shown in Fig. 3B, transient overexpression of CrkL enhanced 32D/EpoR-Wt cell adhesion. In accordance with our previous report (11), coexpression of a dominant negative R-Ras mutant, R-Ras-43N, partially inhibited the CrkL-enhanced adhesion. Similar to R-Ras-43N, Rap1A-17N also exhibited an inhibitory effect on the CrkL-enhanced adhesion. Furthermore, when coexpressed with R-Ras-43N, Rap1A-17N showed an additive inhibitory effect and nearly abrogated the CrkL-enhanced cell adhesion (Fig. 3B). These results thus suggest that Rap1 as well as R-Ras may be involved in CrkL-mediated activation of hematopoietic cell adhesion.

To explore further the possible involvement of Rap1 in integrin activation of hematopoietic cells, we next employed a constitutively activated mutant of Rap1, Rap1A-63E. As shown in Fig. 3C, transient expression of Rap1A-63E in 32D/EpoR-Wt cells induced a dose-dependent activation of cell adhesion to fibronectin. We also established a 32D/EpoR-Wt clone, 32DE/TA-Rap1A-63E, which inducibly expresses Rap1A-63E when withdrawn from tetracycline, and examined the effect of Rap1A-63E on cell adhesion. As shown in Fig. 3D, when 32DE/TA-Rap1A-63E cells were cultured overnight in medium containing various concentrations of tetracycline, the expression of Rap1-63E was induced significantly at tetracycline concentrations equal to or lower than 10 ng/ml. At these concentrations, 32DE/TA-Rap1A-63E cells exhibited significantly increased adhesion to wells coated with 5 µg/ml fibronectin (Fig. 3D). Fig. 3E further demonstrates that 32D/TA-Rap1A-63E cells exhibited significantly enhanced adhesion to wells coated with various concentrations of fibronectin when the expression of Rap1A-63E was induced. As shown in Fig. 3F, the Rap1A-63E-induced as well as basal adhesion of 32DE/TA-Rap1A-63E cells was abrogated by incubation with anti-beta 1 integrin monoclonal antibody, which indicates that Rap1 activates hematopoietic cell adhesion to fibronectin through beta 1 integrins. Fig. 3G further demonstrates that the Rap1A-63E-enhanced cell adhesion was inhibited by anti-alpha 4 and anti-alpha 5 integrin antibodies in an additive way, thus suggesting that both VLA-4 (alpha 4beta 1) and VLA-5 (alpha 5beta 1) are involved in Rap1-induced cell adhesion to fibronectin. Taken together with our previous observation that CrkL-enhanced cell adhesion is mediated by the beta 1 integrins VLA-4 and VLA-5 (11), these data strongly support our hypothesis that Rap1 plays a role in CrkL-mediated activation of hematopoietic cell adhesion.

Rap1 Activation in Hematopoietic Cells Is Also Mediated by the Second Messengers of PLC-gamma -- Recent studies have shown that calcium, diacylglycerol, and cAMP mediate activation of Rap1 by various stimuli in cell type-specific ways (17, 18). Previous studies have shown that the cytokine receptors, including the receptors for Epo (40) and IL-3 (41), activate PLC-gamma , which transduces signals through generation of diacylglycerol as well as through an increase in intracellular calcium. In accordance with this, Epo as well as IL-3 induced tyrosine phosphorylation of PLC-gamma in 32D/EpoR-Wt cells (Fig. 4A). Epo stimulation also induced the physical association of PLC-gamma with the tyrosine-phosphorylated EpoR (Fig. 4A), which agrees with a previous report on UT-7 cells (40). Because PLC-gamma is activated by tyrosine phosphorylation (42), we next examined the possible involvement of the second messengers of PLC-gamma as well as cAMP in cytokine-induced Rap1 activation in hematopoietic cells. As shown in Fig. 4B, treatment of 32D/EpoR-Wt cells with a diacylglycerol analogue, PMA, induced activation of Rap1, which was detectable as early as 1 min after PMA addition and peaked at 5 min. Treatment with a calcium ionophore, ionomycin, similarly induced activation of Rap1, which peaked at 5 min after treatment. On the other hand, forskolin, which activates adenylyl cyclase to generate cAMP, showed no stimulatory activity toward Rap1 in 32D/EpoR-Wt cells. Treatment of cells with a vehicle, dimethyl sulfoxide (Me2SO), alone showed no effect on Rap1 activation (data not shown). Anti-Rap1 blotting of total cell lysates demonstrated that treatment of cells with these reagents had no effect on the expression level of Rap1 (Fig. 4B, lower panel). It was, however, revealed that treatment of cells with forskolin for 20 min induced the appearance of a slower migrating form of Rap1 (Fig. 4B, lower panel), which is in agreement with a previous observation that protein kinase A, activated by cAMP, phosphorylates Rap1 (43). These results suggest that diacylglycerol and calcium, but not cAMP, may be involved in Rap1 activation in 32D cells. Therefore, we next examined the effect of U73122, an inhibitor of PLC, on cytokine-induced activation of Rap1. As shown in Fig. 4C, U73122 drastically decreased Epo- as well as IL-3-induced activation of Rap1. On the other hand, U73343, an inactive analogue of U73122, showed no significant effect on cytokine-induced activation of Rap1 (data not shown). The specificity of the inhibitory effect of U73122 was supported further by the observation that U73122 showed no significant inhibitory effects on Epo-induced activation of Stat5 and Erk (Fig. 4D), which mediate the two major signaling pathways from the EpoR (1). Furthermore, because CrkL plays an important role in Epo-induced activation of the Ras/Erk pathway in 32D/EpoR-Wt cells (10), the absence of inhibitory effect on Erk suggests that U73122 should not affect the CrkL-mediated signaling pathways. In accordance with this, U73122 showed no effects on Epo-induced tyrosine phosphorylation of CrkL and its association with various signaling molecules in 32D/EpoR-Wt cells (data not shown). Together, these results suggest that PLC-gamma may play a crucial role, independently of CrkL, in cytokine-induced activation of Rap1 in hematopoietic cells.


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Fig. 4.   Effects of various pharmacological reagents on Rap1 activation and cell adhesion. A, after being starved overnight from Epo, 32D/EpoR-Wt cells were stimulated with 10 units/ml Epo (E) or 10 ng/ml IL-3 (I), as indicated, for 5 min. Cells were lysed and subjected to immunoprecipitation with anti-PLC-gamma . Immunoprecipitates were resolved by SDS-PAGE and subjected to anti-phosphotyrosine (alpha PY) blotting followed by reprobing with anti-EpoR (alpha EpoR) and anti-PLC-gamma (alpha PLCgamma ), as indicated. Tyrosine-phosphorylated PLC-gamma (PLCgamma -PY) and EpoR (EpoR-PY) are indicated. B, 32D/EpoR-Wt cells were starved from Epo overnight in serum-free medium and incubated with 100 ng/ml PMA, 1 µM ionomycin, or 50 µM forskolin, for various times, as indicated, and subjected to the Rap1 activation assays. Rap1-GTP eluted from GST-RalGDS-RBD (upper panel) and total cell lysates (lower panel) was subjected to immunoblotting with anti-Rap1 antibody. C, after starvation, 32D/EpoR-Wt cells were pretreated with 1 µM U73122 or 0.01% Me2SO for 30 min at 37 °C, as indicated. Cells were stimulated with 10 units/ml Epo or 10 ng/ml IL-3 for the indicated times and subjected to the Rap1 activation assays. D, 32D/EpoR-Wt cells were starved overnight from Epo, pretreated for 30 min at 37 °C with the indicated concentrations of U73122, and stimulated with Epo for 15 min. Cell lysates were subjected to immunoblotting with anti-phospho-Stat5 (alpha Stat5-PY) followed by reprobing with anti-phospho-Erk (alpha Erk-P) and anti-Erk1/2 (alpha Erk), as indicated. E, 32D/EpoR-Wt cells, transiently transfected with pGL-cont., were subjected to the cell adhesion assays in the presence or absence of 100 ng/ml PMA or 1 µM ionomycin, as indicated. F, 32DE/Tet-CrkL cells were cultured overnight in Epo-containing medium with or without 100 ng/ml tetracycline, as indicated. Cells were fluorescently labeled, pretreated for 30 min at 37 °C with 0.1% Me2SO or with 1 µM U73122, as indicated, and subjected to the cell adhesion assays measured by fluorometry as described under "Experimental Procedures." G and H, 32D/EpoR-Wt (G) or 32DE/CrkL (H) cells were fluorescently labeled, pretreated for 30 min at 37 °C with the indicated concentrations of U73122, and subjected to the cell adhesion assays as measured by fluorometry. The values of cell adhesion in the presence of U73122 are expressed relative to that in the absence of U73122. For measurement of Rap1 activities, 32D/EpoR-Wt (G) or 32DE/CrkL (H) cells were starved overnight from Epo, pretreated for 30 min at 37 °C with the indicated concentrations of U73122, stimulated with Epo for 15 min, and subjected to the Rap1 activation assays. Eluates from the GST-RalGDS-RBD precipitates (upper panels) or total cell lysates (lower panels) were subsequently subjected to immunoblotting with anti-Rap1 antibody. Relative Rap1 activities, determined by densitometric analysis, are shown between the two blots. I, 32DE/TA (TA) or 32DE/TA-Rap1A-63E (Rap1A-63E) cells were removed from tetracycline overnight in culture medium with or without 1 unit/ml Epo, as indicated. Cells were fluorescently labeled, pretreated for 30 min at 37 °C with 0.1% Me2SO or 1 µM U73122, as indicated, and subjected to the cell adhesion assays as measured by fluorometry.

Next, the effects of these chemical reagents on cell adhesion were examined. Fig. 4E demonstrates that PMA treatment dramatically increases the adhesion of 32D/EpoR-Wt cells to fibronectin. Treatment with ionomycin also increased, though modestly, the cell adhesion in repeated experiments (Fig. 4E and data not shown). In contrast, U73122 drastically inhibited the adhesion of 32DE/Tet-CrkL cells in the presence of tetracycline (Fig. 4F). U73122, however, showed only a modest inhibitory effect on the 32DE/Tet-CrkL cell adhesion which was enhanced remarkably by overexpression of CrkL in the absence of tetracycline (Fig. 4F). The inhibitory effects of U73122 on activation of Rap1 and cell adhesion were examined further in 32D/EpoR-Wt as well as in 32DE/CrkL, a clone of 32D/EpoR-Wt which constitutively overexpresses CrkL. As shown in Fig. 4G, the Epo-induced Rap1 activation as well as cell adhesion was inhibited significantly by U73122 at a concentration as low as 0.01 µM in 32D/EpoR-Wt cells and was decreased at 1 µM to about one-fifth of that observed in the absence of U73122. On the other hand, the Epo-induced Rap1 activation as well as cell adhesion of 32DE/CrkL cells was inhibited significantly by U73122 only at a concentration as high as 1 µM (Fig. 4H). Therefore, the effects of these pharmacological reagents on the cell adhesion correlated with their effects on Rap1 activation in 32D/EpoR-Wt clones, which supports the idea that Rap1 plays an important role in activation of hematopoietic cell adhesion. In addition, it was suggested that CrkL mediates the Epo-induced Rap1 activation independently of PLC-gamma because the inhibitory effect of U73122 on Epo-induced Rap1 activation in cells overexpressing CrkL was much less significant than that in parental cells.

We demonstrated previously that 32DE/Tet-CrkL cells overexpressing CrkL, but not 32DE/TA cells, significantly adhere to fibronectin-coated wells even when starved from cytokines (11). In the absence of any cytokines, 32DE/TA-Rap1A-63E cells inducibly expressing Rap1A-63E also showed a significant adhesion (Fig. 4I). Importantly, U73122 did not show any significant inhibitory effect on the adhesion of 32DE/TA-Rap1A-63E cells in the absence of cytokines, although the Epo-augmented adhesion of Rap1E-expressing cells was partly inhibited by the PLC inhibitor (Fig. 4I). These results thus agree with our hypothesis that Rap1 is activated downstream of PLC-gamma as well as by the CrkL/C3G signaling pathway and activates integrin-mediated hematopoietic cell adhesion.

Involvement of CrkL and Rap1 in Signaling from the Endogenous EpoR-- Because the 32D/EpoR-Wt cells heterologously express the EpoR, we next examined the human leukemic UT-7 cell line to confirm the involvement of CrkL and Rap1 in signaling from the endogenously expressed EpoR. As shown in Fig. 5,A and B, Epo induced the time- and dose-dependent tyrosine phosphorylation of CrkL in UT-7 cells. The UT-7 cell adhesion to fibronectin-coated wells in the presence of Epo was enhanced significantly by transient overexpression of CrkL or an activated mutant of C3G, C3G-dN, and was inhibited by that of a dominant negative C3G mutant, C3G-dSS (Fig. 5C). U73122 also inhibited the UT-7 cell adhesion in the presence of Epo (Fig. 5D). These results indicate that CrkL is involved in signaling from the endogenous EpoR and, along with C3G, plays a role in activation of adhesion of UT-7 cells. In addition, the inhibitory effect of U73122 indicates that PLC-gamma , activated by Epo in UT-7 cells (40), may also play a role in Epo-induced activation of UT-7 cell adhesion.


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Fig. 5.   Involvement of Rap1 and CrkL in signaling from the endogenous EpoR. A and B, UT-7 cells were starved from Epo overnight and stimulated with 10 units/ml Epo for the indicated times (A) or with the indicated concentrations of Epo for 15 min (B). Cell lysates were immunoprecipitated with anti-CrkL and subjected to anti-phosphotyrosine (alpha PY) immunoblotting followed by reprobing with anti-CrkL (alpha CrkL), as indicated. C, UT-7 cells were transfected with 50 µg of pSG-CrkL (CrkL), pcDNA-C3G-dN (C3G-dN), or pcDNA-C3G-dSS (C3G-dSS), as indicated, along with 1 µg of pGL-cont. Transiently transfected cells were subjected to the cell adhesion assays measured by the luciferase assay as described under "Experimental Procedures." D, UT-7 cells cultured in Epo-containing medium were fluorescently labeled, pretreated for 30 min at 37 °C with 0.1% Me2SO or with 1 µM U73122, as indicated, and subjected to the cell adhesion assays as measured by fluorometry. E and F, UT-7 cells, starved overnight from Epo in serum-free medium, were stimulated with 10 units/ml Epo for indicated times (E) or with the indicated concentrations of Epo for 15 min (F), and subjected to the Rap1 activation assays. Eluates from the GST-RalGDS-RBD precipitates (upper panels) or total cell lysates (lower panels) were probed with anti-Rap1. Relative Rap1 activities are shown between the two anti-Rap1 blots. G and H, UT-7 cells were starved overnight from Epo and fluorescently labeled. Cells were left unstimulated as a control (C) or stimulated with 10 units/ml Epo for indicated times (G) or with the indicated concentrations of Epo for 30 min (H) before being allowed to adhere to fibronectin-coated wells for 30 min for the cell adhesion assays as measured by fluorometry.

It was also confirmed that Epo activates Rap1 in UT-7 cells (Fig. 5,E and F). The Epo-induced activation of Rap1, conspicuously observed when cells were stimulated with as low as 0.1 unit/ml Epo, peaked at 5-15 min after Epo stimulation and gradually declined thereafter. We also examined the effect of Epo on the adhesion of UT-7 cells starved overnight from Epo. As shown in Fig. 5G, Epo modestly increased the adhesion of cytokine-starved UT-7 cells even when added immediately before cells were allowed to adhere to fibronectin-coated wells for 30 min. The effect of Epo on cell adhesion peaked at the preincubation period of 30 min and declined slightly at 60 min. We also examined the dose effect of Epo on UT-7 cell adhesion. As shown in Fig. 5H, the cell adhesion was stimulated significantly by Epo at a concentration as low as 0.1 unit/ml. Because Rap1 as well as cell adhesion was activated significantly by Epo at a concentration as low as 0.1 unit/ml and within 30 min of stimulation, these observations in UT-7 cells are in accordance with our hypothesis that Epo stimulates cell adhesion through activation of Rap1.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present studies have demonstrated that IL-3 and Epo induce Rap1 activation in a hematopoietic cell line, 32D/EpoR-Wt. We have found previously that CrkL forms a stable complex with C3G in hematopoietic cells and is recruited to the activated cytokine receptor complex through interaction between the CrkL SH2 domain and tyrosine-phosphorylated signaling molecules, including Cbl, Shc, and SHP-2 (9). Our previous studies have demonstrated further that the CrkL-C3G complex is involved in cytokine-induced activation of Ras (10). Because C3G activates Rap1 more efficiently than Ras, it is plausible to hypothesize that the recruitment of the CrkL-C3G complex to the cytoplasmic membrane is involved in cytokine-induced activation of Rap1 as well as that of Ras. In accordance with this hypothesis, the cytokine-stimulated Rap1 activation was enhanced by the induced overexpression of CrkL or C3G in 32DE/Tet-CrkL or 32DE/Tet-C3G cells, respectively (Fig. 2). Recently, the CrkL-C3G complex has also been implicated in Rap1 activation in cells stimulated with other growth factors and cytokines as described in the Introduction.

Rap1 was also activated in 32D/EpoR-Wt cells treated with PMA or a calcium ionophore, ionomycin, but the cytokine-induced Rap1 activation was inhibited by a PLC inhibitor, U73122. It is thus hypothesized that PLC-gamma , which Epo as well as IL-3 activates in hematopoietic cells (40, 41), also mediates Rap1 activation through its second messengers, diacylglycerol and calcium. The strong inhibitory effects of U73122 on cytokine-induced activation of Rap1 and cell adhesion further imply that the PLC-mediated pathway may play a more important role than that involving the CrkL-C3G complex in the Epo- or IL-3-induced activation of Rap1 and cell adhesion. It should be noted, however, that dominant negative mutants of CrkL and C3G significantly inhibited cytokine-induced cell adhesion in our previous report (11). In addition, because U73122 exhibited less profound inhibitory effects on activation of Rap1 and cell adhesion in cells overexpressing CrkL, it is speculated that CrkL mediates the activation of Rap1 and cell adhesion independently of PLC-gamma . Recent studies have also shown that PLC plays a critical role in Rap1 activation induced by the B cell receptor stimulation in B lymphocytes and that induced by thrombin in platelets (20, 21). CalDAG-GEF-I (23), a Rap1 GEF that is highly expressed in hematopoietic cells and is activated by diacylglycerol or calcium, has been implicated in PLC-mediated activation of Rap1. A recent study in platelets (44), however, has demonstrated that protein kinase C, which is activated by diacylglycerol, may also be involved in a delayed and sustained second phase of Rap1 activation, which is observed following a very rapid and transient activation mediated by calcium in platelets stimulated with thrombin. Because the cytokine-induced Rap1 activation in hematopoietic cells we observed in this study is also sustained, it is possible that protein kinase C may be involved in its activation. It should be also mentioned that an intracellular calcium chelator, BAPTA-AM, showed no inhibitory effect on Epo- or IL-3-induced activation of Rap1 in 32D cells,2which is in contrast to previous reports on PLC-dependent Rap1 activation observed rapidly and transiently following thrombin stimulation in platelets or that observed in lymphocytes stimulated though the B cell antigen receptor (20, 21). It is thus possible that, although Epo stimulation induces an increase in intracellular calcium (45), it may not play a critical role in Rap1 activation. Further studies are required to elucidate the downstream signaling pathways mediating the PLC-gamma -dependent Rap1 activation in cytokine-stimulated hematopoietic cells.

Tsukamoto et al. (46) reported previously that, when cultured in G-CSF, the Rap1 activity increased gradually over several days in a subclone of 32D cells which undergoes granulocytic differentiation with concomitant increases in expression of various adhesion molecules, such as VLA-4, LFA-1, and CD44, and in cell adhesiveness under the same condition. Tsukamoto et al. (46) further demonstrated that overexpression of SPA-1, a Rap1-specific GTPase-activating protein, drastically inhibited the activation of Rap1 and adhesion of 32D cells without affecting the G-CSF-induced morphological differentiation and augmentation in expression of adhesion molecules. It was thus speculated that Rap1-GTP may be required for the adhesion of 32D cells cultured in G-CSF. However, the adhesion molecules involved in adhesion of differentiated 32D cells induced by G-CSF were not examined. Furthermore, because the G-CSF-induced increase in Rap1 took place very gradually over a very long period of time (46), it has remained to be known whether Rap1 is involved directly in signaling mediated through the G-CSF receptor or in inside-out signaling mediating G-CSF-induced integrin activation.

Several observations in the present studies, on the other hand, indicate that Rap1 plays a critical role in cytokine-induced activation of hematopoietic cell adhesion mediated through beta 1 integrins. First, the basal as well as cytokine-stimulated Rap1 activity was increased by the induction of CrkL or C3G overexpression in 32D cells (Fig. 2), which also enhanced the basal as well as cytokine-stimulated adhesion of cells through VLA-4 and VLA-5 as demonstrated previously (11). The Rap1 activity, thus, correlated with the beta 1 integrin-mediated cell adhesiveness in these cells. Second, the correlation was also observed in cells treated with various chemical reagents, including U73122, which drastically inhibited both cytokine-induced Rap1 activation and cell adhesion. (Fig. 4). Last, a dominant negative mutant of Rap1, Rap1A-17N, inhibited CrkL-enhanced adhesion of hematopoietic cells, whereas an activated Rap1 mutant, Rap1A-63E, enhanced cytokine-induced cell adhesion or induced adhesion of cells starved from cytokines (Fig. 3). It should be also noted that the cell adhesion induced by overexpression of CrkL and that induced by expression of Rap1A-63E are both mediated through the beta 1 integrins VLA-4 and VLA-5, as demonstrated by inhibitory effects of specific antibodies. These observations are in agreement with the idea that the CrkL-C3G complex mediates cytokine-induced integrin activation, at least partly, by activating Rap1. R-Ras may also be involved in this process because a dominant negative mutant of R-Ras, R-Ras-43N, also showed an inhibitory effect on CrkL-enhanced cell adhesion in an additive way with Rap1A-17N (Fig. 3B). This is in accordance with previous reports that R-Ras activates integrin in 32D cells (32). Previous reports have also implicated PLC-gamma in inside-out signaling to activate beta 1 integrins in hematopoietic cells (47, 48), which is consistent with our observation that U73122 inhibited cytokine-induced cell adhesion (Fig. 4). In contrast, U73122 failed to inhibit cytokine-independent adhesion of cells expressing Rap1A-63E, which is in agreement with the notion that Rap1 functions downstream of PLC-gamma to mediate the cytokine-induced integrin activation signal. Together, these results support the hypothesis that the receptors for Epo and IL-3 transduce inside-out signaling to activate beta 1 integrins by stimulating Rap1 through the CrkL/C3G and PLC-gamma pathways.

While this manuscript was in preparation, Katagiri et al. (49) reported that an activated Rap1 mutant, Rap1-12V, stimulates adhesion of a murine pro-B-cell line, BA/F3, through heterologously expressed human LFA-1, a beta 2 integrin, to ICAM-1 and further suggested that Rap1 may be involved in T cell receptor-mediated activation of LFA-1. Furthermore, Reedquist et al. (50) reported that Rap1-12V stimulates T lymphocyte adhesion through VLA-4 to VCAM-1 as well as through LFA-1 to ICAM-1. Reedquist et al. (50) further demonstrated CD31-dependent activation of Rap1 and its involvement in CD31-dependent cell adhesion. Whereas Reedquist et al. (50) suggested, by using an activation-specific antibody, that Rap1 may not directly increase the ligand binding affinity of LFA-1, Katagiri et al. (49) showed that Rap1 increases the affinity of LFA-1 for ICAM-1. It is thus possible that Rap1 may activate beta 2 integrin-mediated cell adhesion through different mechanisms in different cell types. On the other hand, the mechanism by which Rap1 stimulates hematopoietic cell adhesion through beta 1 integrins as well as the downstream signaling events mediating this process has remained to be examined. Irrespective of the mechanisms, our study and these reports collectively indicate that Rap1 is involved in inside-out signaling to activate both beta 1 and beta 2 integrins in hematopoietic or lymphoid cells in response to various stimuli.

    ACKNOWLEDGEMENTS

We are grateful to Drs. John Groffen, Erkki Ruoslahti, Michiyuki Matsuda, Makoto Noda, Masakazu Hattori, Johannes L. Bos, and Norio Komatsu for the generous gifts of experimental materials. We also thank Drs. Masayuki Yoshida and Toshihiko Iizuka for assistance in fluorometric adhesion assays, Drs. Yoji Ikawa and Masakazu Hattori for helpful discussions, and Eiko Nishimura and Kaori Okada for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan and the Ichiro Kanehara Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-5803-5952; Fax: 81-3-5803-0131; E-mail: miura.med1@med.tmd.ac.jp.

Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M004627200

2 A. Arai, Y. Nosaka, E. Kanda, and O. Miura, unpublished results.

    ABBREVIATIONS

The abbreviations used are: Epo, erythropoietin; EpoR, Epo receptor; IL, interleukin; Stat, signal transducers and activators of transcription; Erk, extracellular signal-regulated kinase; PLC, phospholipase C; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; SH, Src homology; GEF, guanine nucleotide exchange factor; Tet, tetracycline; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; RBD, Rap1 binding domain; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; ICAM, intercellular adhesion molecule; VCAM, vascular cellular adhesion molecule; XTT, 3'-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate; LFA-1, lymphocyte function-associated antigen-1.

    REFERENCES
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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