The α-Chemokine, Stromal Cell-derived Factor-1α, Binds to the Transmembrane G-protein-coupled CXCR-4 Receptor and Activates Multiple Signal Transduction Pathways*

The α-chemokine stromal cell-derived factor (SDF)-1α binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation. The signaling pathways that mediate the effects of SDF-1α are not well characterized. We studied events following SDF-1α binding to CXCR-4 in a model murine pre-B cell line transfected with human CXCR-4. There was enhanced tyrosine phosphorylation and association of components of focal adhesion complexes such as the related adhesion focal tyrosine kinase, paxillin, and Crk. We also observed activation of phosphatidylinositol 3-kinase. Wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase, partially inhibited the SDF-1α-induced migration and tyrosine phosphorylation of paxillin. SDF-1α treatment selectively activated p44/42 mitogen-activated protein kinase (Erk 1 and Erk 2) and its upstream kinase mitogen-activated protein kinase kinase but not p38 mitogen-activated protein kinase, c-Jun amino-terminal kinase or mitogen activated protein kinase kinase. We also observed that SDF-1α treatment increased NF-κB activity in nuclear extracts from the CXCR-4 transfectants. Taken together, these studies revealed that SDF-1α activates distinct signaling pathways that may mediate cell growth, migration, and transcriptional activation.

The ␣-chemokine stromal cell-derived factor (SDF)-1␣ binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation. The signaling pathways that mediate the effects of SDF-1␣ are not well characterized. We studied events following SDF-1␣ binding to CXCR-4 in a model murine pre-B cell line transfected with human CXCR-4. There was enhanced tyrosine phosphorylation and association of components of focal adhesion complexes such as the related adhesion focal tyrosine kinase, paxillin, and Crk. We also observed activation of phosphatidylinositol 3-kinase. Wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase, partially inhibited the SDF-1␣-induced migration and tyrosine phosphorylation of paxillin. SDF-1␣ treatment selectively activated p44/42 mitogen-activated protein kinase (Erk 1 and Erk 2) and its upstream kinase mitogenactivated protein kinase kinase but not p38 mitogenactivated protein kinase, c-Jun amino-terminal kinase or mitogen activated protein kinase kinase. We also observed that SDF-1␣ treatment increased NF-B activity in nuclear extracts from the CXCR-4 transfectants. Taken together, these studies revealed that SDF-1␣ activates distinct signaling pathways that may mediate cell growth, migration, and transcriptional activation.
Despite the increasingly prominent role of SDF-1␣ and its receptor CXCR-4 in the regulation of cell proliferation, migration, and HIV infection, relatively little is known about the signaling pathways that may mediate these effects (19,20). In this study, we show that SDF-1␣ stimulation in CXCR-4 transfectants results in the increased phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK/Pyk2), Crk, and paxillin. SDF-1␣ treatment activated the p44/42 MAP kinases (Erk 1 and 2), PI-3 kinase, and NF-B. These studies indicate that activation of CXCR-4 results in modulation of signaling molecules and transcription factors that mediate changes in the cytoskeletal apparatus and also regulate cell growth.

EXPERIMENTAL PROCEDURES
Reagents and Materials-RAFTK antibodies were generated using C domain glutathione S-transferase fusion proteins as described previously (29). Serum R-4250 was chosen for further studies based on its titer in enzyme-linked immunosorbent assay. This antiserum does not cross-react with FAK and recognizes both human and murine forms of RAFTK. Monoclonal anti-phosphotyrosine antibody (4G10) was a generous gift from Dr. Brian Druker (Oregon Health Sciences University, Portland, OR). Purified antibodies to JNK, p38 MAP kinase, p44/42 MAPK, and recombinant GST-c-Jun amino-terminal proteins (1-79 amino acids) were obtained from Santa Cruz Laboratories (Santa Cruz, CA). Antibodies to paxillin and Crk were obtained from Transduction Laboratories, Inc. (Lexington, KY). Monoclonal antibodies to CXCR-4 and the isotype control were from PharMingen (San Diego, CA). Electrophoresis reagents were obtained from Bio-Rad. The protease inhibitors leupeptin and ␣ 1 -antitrypsin as well as all other reagents were obtained from Sigma. Wortmannin was obtained from Calbiochem, and the nitrocellulose membrane was from Bio-Rad. Indo-1 acetoxymethyl ester (Indo-1 AM) was purchased from Molecular Probes (Eugene, OR).
Construction of CXCR-4 Stable Transfectants-We used a murine pre-B lymphoma cell line, L1.2, for the transfection studies. CXCR-4 cDNA, tagged at the amino terminus with a Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), was subcloned into the pcDNAIII expression vector. The DNA was stably transfected into the L1.2 cells as described (30 -32), and G418-selective medium was used to select for transfectants. Cell-surface expression of CXCR-4 on the transfectants was confirmed by FACS analysis.
FACS Analysis-The CXCR-4 L1.2 transfectants (1 ϫ 10 6 ) were washed twice with phosphate-buffered saline (PBS), resuspended in 100 l of PBS containing 5% FCS and 5 g/ml PE-labeled CXCR-4 antibody or isotype control (PharMingen), and then incubated for 30 min at 4°C. The cells were washed twice with ice-cold PBS, 5% FCS, resuspended in 500 l of PBS, 5% FCS buffer, and then analyzed by flow cytometry to determine the levels of surface expression of these receptors.
Calcium Flux Assay-The CXCR-4 transfectants were washed with RPMI 1640 and resuspended at 10 ϫ 10 6 cells/ml in the RPMI medium. The cells were loaded with Indo-1 AM (Molecular Probes) by adding 5 l of working Indo-1 solution to the 10 ϫ 10 6 cells that were suspended in 1 ml of RPMI solution and incubated for 45 min at 37°C. Cells were diluted to 1 ϫ 10 6 /ml, treated with SDF-1␣, and analyzed for calcium mobilization by flow cytometry (Coulter Electronics, Hialeah, FL) as described (33). Calcium flux assays and all other subsequent signaling assays were repeated at least three times.
Stimulation of Cells-Cells were washed twice with RPMI 1640 (Life Technologies, Inc.) and resuspended at 10 ϫ 10 6 cells/ml in the same medium. Cells were starved for 4 h at 37°C and then stimulated with different concentrations of SDF-1␣ at 37°C for various periods. After stimulation, cells were lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, leupeptin, and pepstatin, 10 mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate). Total cell lysates (TCL) were clarified by centrifugation at 10,000 ϫ g for 10 min. Protein concentrations were determined by protein assay (Bio-Rad). Cell lysis, immunoprecipitation, immunoblotting, kinase assays, and autophosphorylation assays were carried out as described below.
Immunoprecipitation and Western Blot Analysis-For the immunoprecipitation studies, identical amounts of protein from each sample were clarified by incubation with protein A-Sepharose CL-4B or Gammabind plus Sepharose (Amersham Pharmacia Biotech) for 1 h at 4°C. The beads were removed by brief centrifugation, and the solution was incubated with different primary antibodies for each experiment for 4 h or overnight at 4°C. Antibody-antigen complexes were immunoprecipitated by incubation with 50 l of protein A-Sepharose or Gammabind Sepharose (10% suspension) for 4 h at 4°C. The Sepharose beads were washed three times with modified RIPA buffer and one time with PBS to remove the nonspecifically bound proteins. Bound proteins were solubilized in 40 l of 2ϫ Laemmli buffer and further analyzed by immunoblotting. The samples were separated on SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked and probed with primary antibody for 3 h at room temperature (RT) or 4°C overnight. Immunoreactive bands were visualized using horserad-ish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent (ECL) system (Amersham Pharmacia Biotech). Monoclonal antibody (4G10, IgG2a) was used for Western blot analysis of the phosphotyrosine protein.
RAFTK Kinase Assays-In vitro kinase assays were performed as described earlier (34). The cell lysates immunoprecipitated with RAFTK antiserum were washed twice with RIPA buffer and once in kinase buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl 2 , 5 mM MnCl 2 , 100 mM Na 3 VO 4 ). For the in vitro kinase assays, the immune complex was incubated in kinase buffer containing 25 g of poly(Glu: Tyr) (4:1), 20 -50 kDa (Sigma), and 5 Ci of [␥-32 P]ATP at RT for 30 min. The reaction was stopped by adding 2ϫ SDS sample buffer and boiling the sample for 5 min at 100°C. Proteins were then separated on 10% SDS-PAGE and detected by autoradiography. Normal rabbit serum was used as a negative control. The autophosphorylation assay was carried out by incubating the immune complex in kinase buffer containing 5 Ci of [␥-32 P]ATP at RT for 30 min. The reaction was stopped by adding 4ϫ SDS sample buffer and by boiling the sample for 5 min. Proteins were then separated on SDS-PAGE and detected by autoradiography.
JNK, p44/42 MAP Kinase, and p38 MAP Kinase Assays-The JNK assay was performed as described earlier (35). Briefly, cell lysates were immunoprecipitated with JNK antibody (Santa Cruz Biotechnology).  (0) or cells stimulated with SDF-1␣ (100 ng/ml) for the indicated times were immunoprecipitated with RAFTK antibody or normal rabbit serum as the control. A, the immune complexes were resolved on 7.5% SDS-PAGE gels and subjected to serial immunoblotting with anti-phosphotyrosine antibody (top panel) and RAFTK antibody (bottom panel). B, the immunoprecipitates were subjected to autokinase assay. C, the immune complexes were subjected to in vitro kinase assays using poly-(Glu:Tyr), 4:1, as a substrate. The 32 P-incorporated proteins were resolved on 10% SDS-PAGE, followed by autoradiography. C, control.
The immune complexes were washed twice with RIPA buffer and once in kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl 2 , 20 M ATP). The complex was then incubated in kinase buffer containing recombinant GST-c-Jun 0.2 g/l (1-79 amino acids) (Santa Cruz Biotechnology) and 5 Ci of [␥-32 P]ATP for 10 min at RT. The reaction was terminated by adding 2ϫ SDS sample buffer and boiling the sample for 5 min at 100°C. Proteins were separated on 12% SDS-PAGE and detected by autoradiography. Rabbit IgG was used as a negative control. For the p44/42 and p38 MAP kinase assays, cell lysates from unstimulated or stimulated cells were immunoprecipitated with Erk 1 and Erk 2 (p44/ 42) or p38 MAP kinase antibody (Santa Cruz Biotechnology). The immune complexes were washed twice with RIPA buffer and once in kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl 2 and 20 M ATP). The complex was then incubated in kinase buffer containing 7 g of myelin basic protein (Upstate Biotechnology, Lake Placid, NY) and 5 Ci of [␥-32 P]ATP for 20 min at 30°C. Proteins were separated on 15% SDS-PAGE and detected by autoradiography. Rabbit IgG was used as a negative control. SDS-PAGE and detected by autoradiography. Rabbit IgG was used as a negative control.

MKK-1 and MKK-4 Kinase Assays-For
In Vitro PI-3 Kinase Assay-PI-3 kinase assays were performed as described (36). Briefly, equal amounts of protein from each sample were immunoprecipitated with either anti-phosphotyrosine antibody (4G10) or class-matched mouse IgG. Nonspecific binding was removed by washing the samples three times each with PBS containing 1% Nonidet P-40, followed by 0.5 mM Tris containing 0.5 mM lithium chloride and then by TE buffer. Samples were resuspended in 20 l of TE buffer, 20 l of phosphoinositol (10 g, Avanti Polar Lipids, Alabaster, AL), and 10 l of ATP mix (1 mM HEPES, 10 M ATP, 1 M MgCl 2 , and 5 Ci of [␥-32 P]ATP) and incubated at RT for 10 min. The reaction was stopped by adding 60 l of 2 mM HCl and 160 l of chloroform:methanol (1:1 v/v). Lipids were separated on oxalate-impregnated silica TLC plates using a solvent system of chloroform:methanol:water:ammonium hydroxide (20%) (35:35:3.5:7), followed by autoradiography at Ϫ80°C.
Chemotaxis Assay-The chemotaxis assay was performed in 24-well plates containing 5-m porosity inserts (Costar Corp., Kennebunk, ME). Cells grown in RPMI 1640 medium containing 10% FCS were washed twice and suspended as 10 ϫ 10 6 cells per ml in RPMI 1640 and H199 medium (1:1) containing 0.5% bovine serum albumin. Chemokines were then added to the bottom wells, and 100 l (1 ϫ 10 6 ) of cells were loaded onto the inserts. Cells migrating to the bottom well were collected after 2-4 h and counted on a flow cytometer. To assess the effect of wortmannin on migration, the cells were resuspended in medium containing different concentrations of wortmannin, and the chemotaxis assays were done as described above.
Electrophoretic Mobility Shift Assay-Double-stranded oligonucleotides containing the consensus binding site for NF-B (5Ј-AGT TGA GGG GAC TTT CCC AGG C-3Ј) were labeled with [␥-32 P]ATP (3,000 Ci/mmol, NEN Life Science Products) using polynucleotide kinase (Promega, Madison, WI) according to established procedures. 10 g of nuclear extract were incubated with labeled DNA (0.4 ng, 4,400 cpm) for 10 min at RT in the presence of DNA-binding buffer and 250 ng of poly(dI-dC) oligomer (Boehringer Mannheim) as described previously (37). The complexes were then separated on a 7.5% polyacrylamide gel and autoradiographed. The results shown are representative of findings from three independent experiments.

SDF-1␣ Treatment Induces Ca 2ϩ Flux in CXCR-4 L1.2
Transfectants-Human CXCR-4 cDNA was stably transfected into the murine pre-B lymphoma cell line, L1.2. Untransfected and transfected cell lines were analyzed for CXCR-4 expression. As shown in Fig. 1, CXCR-4 transfectants expressed high levels of the receptor in these transfected cells. Signal transduction by the binding of ligands to their cognate chemokine receptors involves characteristic calcium fluxes. To confirm that the CXCR-4 L1.2 cells expressing functional human CXCR-4 receptors retained this fundamental signaling property, the cells were treated with SDF-1␣, and calcium fluxes were monitored by FACS analysis. SDF-1␣ treatment induced characteristic calcium fluxes in the CXCR-4 L1.2 cells (data not shown).
SDF-1␣ Treatment Activates RAFTK-RAFTK, a recently identified member of the focal adhesion kinase family, has been shown to be activated by various growth factors and chemokines (34, 38 -40). We therefore investigated whether SDF-1␣ activates RAFTK in L1.2 transfectants. We observed rapid phosphorylation of endogenous murine RAFTK in the transfected L1.2 cells upon SDF-1␣ stimulation ( Fig. 2A). We also observed an increase in the intrinsic tyrosine kinase activity of RAFTK following SDF-1␣ treatment, as determined by an autophosphorylation assay and in vitro kinase assay in which poly(Glu:Tyr) (4:1) was used as an exogenous substrate (Fig. 2,  B and C). ation state of these proteins. As shown in Fig. 3, A and B, SDF-1␣ stimulation resulted in enhanced tyrosine phosphorylation of paxillin and Crk. Equivalent amounts of these proteins were present in each lane (bottom panels).

SDF-1␣ Treatment Induces Tyrosine Phosphorylation and Association of Focal Adhesion Components
It has been shown that upon activation by cytokines the adaptor molecule Crk associates with other components of focal adhesions to enhance signaling (41)(42)(43). We therefore investigated whether SDF-1␣ treatment results in changes in the association of Crk with paxillin and RAFTK. As shown in Fig.  4, A and B, Crk associates with paxillin and RAFTK, and this association was enhanced upon SDF-1␣ treatment.
SDF-1␣ Activation Stimulates the MAP Kinase Pathway-It has previously been shown that RAFTK acts upstream of MAP kinase and the JNK pathway (39,47). Recently, we have also shown that the ␤-chemokine, MIP-1␤, stimulated JNK kinase in human CCR5 L1.2 transfectants (33). We further showed that RAFTK mediates activation of JNK in these cells. Fig. 6A shows that SDF-1␣ treatment of CXCR-4 L1.2 cells resulted in FIG. 5. SDF-1␣ stimulation induces PI-3 kinase activity, and its inhibition by wortmannin inhibits migration and tyrosine phosphorylation of paxillin. A, cell lysates (1 mg) untreated or treated with SDF-1␣ (100 ng/ml) were immunoprecipitated with antiphosphotyrosine antibody. The immunocomplexes were washed and subjected to in vitro PI-3 kinase assays as described under "Experimental Procedures." The C lane represents the immunoprecipitates with purified IgG as a control. B, CXCR-4 L1.2 cells untreated or treated with different concentrations of SDF-1␣ were subjected to chemotactic assays as described under "Experimental Procedures." C, the CXCR-4 L1.2 cells were pretreated with 5, 50, or 500 nM concentrations of wortmannin for 30 min and subjected to chemotactic assay in the presence of SDF-1␣ (100 ng/ml) as described under "Experimental Procedures." D, the lysates from unstimulated or SDF-1␣-stimulated cells, in the absence or presence of 100 nM wortmannin, were immunoprecipitated with anti-paxillin antibody. The immune complexes were resolved by 8% SDS-PAGE and subjected to serial immunoblotting with anti-phosphotyrosine antibody (top panel) and anti-paxillin antibody (bottom panel).
the rapid activation of p44/42 MAP kinase. However, no significant effect on JNK or p38 MAP kinase was observed (Fig. 6, B  and C). Furthermore MKK-1, which acts upstream of p44/42 MAP kinase, was activated whereas MKK-4, which acts upstream of JNK, was not altered in response to SDF-1␣ treatment (Fig. 7, A and B). Wortmannin had no effect on SDF-1␣mediated p44/42 MAP kinase activation (data not shown).
SDF-1␣ Modulates NF-B in CXCR-4 L1.2 Transfectants-Various inflammatory and growth-promoting cytokines activate NF-B activity, which is known to regulate transcription of growth-promoting host genes as well as HIV proviral genes via the long terminal repeat element (48,49). To determine whether chemokines may modulate its activity, the binding activity of the NF-B target sequence was tested with electrophoretic mobility shift assay using nuclear proteins prepared from SDF-1␣-treated cells. As shown in Fig. 8, SDF-1␣ treat-ment increased the binding activity of NF-B target sequences. The observed binding activity was specific since the 10ϫ nonradiolabeled NF-B sequence completely competed binding activity, whereas the 10ϫ nonspecific DNA sequence did not change the binding activity of NF-B.

DISCUSSION
The chemokine receptor CXCR-4 and its cognate ligand SDF-1␣ have recently gained considerable interest because of their role in HIV pathogenesis and hematopoietic progenitor migration (11,12,15,16). Furthermore, SDF-1␣ has been shown to be essential for B cell lymphocyte development, since SDF-1␣ null mice have a major defect in both fetal liver and bone marrow B cell lymphopoiesis (11). However, relatively little is known about the signaling pathways mediated by CXCR-4 upon binding of its ligand SDF-1␣ or of HIV envelope proteins (19,20). In this study, we have used human CXCR-4transfected murine pre-B lymphoma L1.2 cells as a model to investigate the signal transduction pathways mediated by the CXCR-4 receptor upon binding to its cognate ligand, SDF-1␣.
Chemokines have been shown to affect chemotaxis which plays a key role in a variety of cell responses including development, wound repair, inflammation, and metastasis (1-7). Coordinated regulation of multiple steps including adhesion and cytoskeleton modification is required for these processes. Various cytokines that induce chemotaxis modulate the formation and function of focal adhesions (50). These adhesions are cytoskeletal structures that form adherent contacts with the extracellular matrix. Changes in the structure of the actin cytoskeleton have been shown to be associated with phosphorylation of focal adhesion components. In the present study, SDF-1␣ enhanced the phosphorylation and association of proteins involved in the formation of focal adhesions. These pro- teins included RAFTK, Crk, and paxillin. RAFTK, also known as Pyk2 or Cak-␤, has been shown to play important roles in various signal transduction pathways (20, 33, 34, 38 -40). RAFTK has been shown to be phosphorylated by ␣and ␤-chemokines and HIV-1 envelope glycoproteins from T-tropic and macrophage-tropic strains (20). Paxillin has also been demonstrated to be phosphorylated by the ␤-chemokines, MIP-1␤ and RANTES, and to participate in integrin-mediated signal transduction pathways (33,51). Crk is a docking protein and plays an important role in assembling signaling complexes (41)(42)(43). We have observed enhanced association of Crk with paxillin and RAFTK upon SDF-1␣ stimulation. Phosphorylation of the focal adhesion components RAFTK, paxillin, and Crk and their association with each other may result in the formation of signaling complexes inducing changes in the cytoskeleton that mediate SDF1-␣-triggered chemotaxis.
Prior studies have demonstrated that PI-3 kinase and its metabolic products play an important role in signaling pathways related to chemotaxis (46). We observed an increase in PI-3 kinase activity after SDF-1␣ treatment. Inhibition of PI-3 kinase activity by wortmannin reduced SDF-1␣-induced cell migration and phosphorylation of paxillin. These results suggest that PI-3 kinase and paxillin phosphorylation play important roles in SDF-1␣-induced cell migration. The data also suggest that PI-3 kinase acts upstream in the signal transduction pathway leading to the tyrosine phosphorylation of paxillin. PI-3 kinase activity has previously been shown to be important for the tyrosine phosphorylation of paxillin mediated by platelet-derived growth factor (52). However, integrin-induced tyrosine phosphorylation of paxillin does not appear to require PI-3 kinase activity (53).
We also investigated the effects of SDF-1␣ on the downstream pathways that are known to mediate transcriptional activation. We observed that SDF-1␣ selectively activated p44/42 MAP kinase (Erk 1 and 2), but not p38 MAPK or JNK. Interleukin 8, another member of the CXC chemokine family, has been shown to activate both p44/42 and p38 MAPK but not JNK (54,55). Interleukin 8 differs from SDF-1␣ by having the characteristic ELR motif (glutamic acid-leucine-arginine) immediately preceding the first cysteine residue near the amino terminus. ␤-Chemokines activate p38 MAPK and JNK (33). This suggests selective regulation of MAP kinase pathways by different types of ␣and ␤-chemokines. The p44/42 and p38 MAP kinases and JNK kinases are regulated by dual specificity kinases which are specific for each MAPK subgroup, thus allowing for their independent regulation. We found that SDF-1␣ activates MKK-1 (also known as MEK-1), which selectively activates p44/42 (Erk) subgroups (56). However, MKK-4 (also known as SEK-1 or JNKK), which activates both p38 MAPK and JNK but does not activate p44/42 (Erk) subgroups (57, 58), was not activated by SDF-1␣. These data provide further new information on the specificity of chemokine signaling effects.
Our studies suggest that SDF-1␣ can lead to the activation of NF-B, a nuclear transcriptional factor. NF-B activation has been extensively studied in inflammatory and immunoregulatory cells and has been shown to regulate gene expression in lymphocytes in response to antigen and cytokine stimulation (48). NF-B is also an important transcription factor in HIV proviral gene expression (49). Activated NF-B has also been shown to regulate the expression of various chemokines by binding to their promoter regions (59). Recently, NF-B was shown to have anti-cell death functions. NF-B activation by tumor necrosis factor -␣ or ionizing radiation suppresses the signal for apoptosis (60,61). SDF-1␣ induction of NF-B activity could result in enhanced cell proliferation, expression of other chemokine genes, as well as enhanced transcription of HIV gene products. This last point may be relevant to the current consideration of therapeutically using chemokines like SDF-1␣ to inhibit HIV infection.
Our results provide new information on the signal transduction pathways utilized by the ␣-chemokine receptor CXCR-4 and show how SDF-1␣ may act on a molecular level to regulate cell migration and growth. We have shown that SDF-1␣ stimulation induces the tyrosine phosphorylation and association of focal adhesion components RAFTK, paxillin, and Crk which may result in the formation of signaling complexes. It appears that SDF-1␣ stimulation of PI-3 kinase activity is essential for its chemotactic effects. Furthermore, SDF-1␣ selectively activates the p44/42 MAP kinase (Erk) but not the p38 MAP kinase or JNK. These results suggest that specific functions of various chemokines may be regulated by different members of the MAP kinase family.