From the Department of Laboratory Medicine and Pathology,

University of Minnesota Medical School,

Minneapolis, MN 55455

Journal of Biological Chemistry 269:32514 (1994)

The B cell-specific cell surface molecule CD19 plays a role in regulating immunoglobulin (Ig) receptor signaling, and cross-linking CD19 activates several signaling molecules in mature human B cells. In surface Ig negative B cell precursors, a protein tyrosine kinase (PTK)-dependent homotypic aggregation response can be triggered by cross-linking CD19. In the current study, we examined the outcome of PTK-mediated signal transduction following CD19 cross-linking on surface Ig negative and surface Ig positive B cell lines, as well as freshly isolated surface Ig negative B cell precursors. PTK activation resulted in the tyrosine phosphorylation of multiple protein substrates, and peaked at 0.5 to 1 min following CD19 cross-linking in all B-lineage cells examined. One of the tyrosine phosphorylated substrates was identified as the hematopoietic-specific protein Vav, a guanine nucleotide exchange factor that activates the Ras pathway. Evidence consistent with Ras pathway activation was also demonstrated by MEK activation and subsequent phosphorylation of a MAP kinase fusion protein. CD19 cross-linking, sequential immunoprecipitation, and Western blotting revealed that: a) Vav becomes associated with CD19, b) phosphatidylinositol 3-kinase (PI 3-kinase) becomes associated with CD19, and c) PI 3-kinase becomes associated with Vav. No such physical interaction occurred following control IgG1 cross-linking or cross-linking of class I major histocompatability complex cell surface molecules. Coupled with a previous report (Tuveson et al., Science 260:986, 1993), our data supports a model in which CD19 cross-linking induces the formation of a signaling complex that leads to the activation of two pathways involving Ras and PI 3-kinase.

CD19 has been shown to be part of a multimeric protein complex on the cell surface of mature B cells, which includes complement receptor 2 (CD21), TAPA-1 (CD81) and Leu-13 (8,9). A model has been proposed whereby the CD19/CD21 complex may modulate complement-dependent immune responses, and enhance the primary B cell response to antigens (10). In this model, CD21 may serve primarily as a ligand binding subunit, while CD19 could function as a signal-transducing subunit. However, it is possible that CD19 also has a specific ligand that has not yet been identified. The coupling of TAPA-1/CD81 to this multimeric protein complex was shown to be critical for CD19-mediated homotypic aggregation (11).

We have recently shown that cross-linking cell surface CD19 induces the homotypic aggregation of normal B cell precursors (18), indirectly suggesting that a signaling event can be transduced through CD19 on B cell precursors. We also found that the PTK inhibitor herbimycin A inhibits CD19-mediated homotypic aggregation (W.-K. Weng and T. W. LeBien, unpublished observations). This suggests that CD19-mediated homotypic aggregation is PTK- dependent, an observation previously reported by Kansas and Tedder (19).

In the current study, we have analyzed PTK activation following CD19 cross-linking in early human B-lineage cells, and have begun to characterize the substrates which undergo tyrosine phosphorylation. One of the signal transduction molecules selectively expressed in hematopoietic cells is the protein encoded by the vav proto-oncogene (20). Recent studies have shown that Vav is rapidly and transiently phosphorylated on tyrosine, and increases its Ras GTP/GDP exchange activity following T cell and B cell antigen receptor-initiated signal transduction (21,22). We now report that cross-linking CD19 on surface Ig negative B cell precursors and surface Ig positive immature B cells results in the rapid tyrosine phosphorylation of Vav, and a concomitant physical association of Vav with CD19 and PI 3-kinase. We further demonstrate that mitogen-activated protein (MAP) kinase-kinase (also known as MEK) and MAP kinase are activated following cross-linking of CD19. These results demonstrate that stimulation of CD19 can rapidly activate two major signaling pathways, suggesting a crucial signaling role for CD19 in antigen-independent and antigen-dependent stages of early B cell development.

The 95 kDa proto-oncoprotein Vav (20), a putative guanine nucleotide exchange factor, becomes tyrosine phosphorylated following cross-linking of surface Ig (22) or the T cell receptor (21). Data in Fig. 2 revealed considerable overlap in the size of the tyrosine phosphorylated substrates following cross-linking of surface IgM and CD19, including a 95 kDa protein. We therefore examined whether Vav became tyrosine phosphorylated following CD19 cross-linking. 1E8 and BLIN-1 were cross-linked with anti-CD19 or anti-class I MHC, lysed, immunoprecipitated with anti-p-Tyr, and immunoblotted with anti-Vav.

As shown in Fig. 3, Vav was detected in anti-p-Tyr immunoprecipitates following 1 and 5 min of CD19 cross-linking in 1E8 and BLIN-1, whereas anti-p-Tyr immunoprecipitates of class I MHC cross-linked cells gave very weak Vav staining after 5 min in BLIN-1 only. Neither control IgG1 cross-linking nor control IgG1 immunoprecipitation revealed any detectable Vav. This result suggests that cross-linking CD19 leads to PTK activation and subsequent tyrosine phosphorylation of Vav. Alternatively, Vav may associate with some tyrosine phosphorylated protein following CD19 cross-linking, and be co-precipitated with anti-p-Tyr mAb. To distinguish between these two possibilities, anti-Vav immunoprecipitates of cell lysates from CD19 cross-linked 1E8 and BLIN-1 were analyzed by anti-p-Tyr immunoblotting. As shown in Fig. 4A, an increase in tyrosine phosphorylation of the 95 kDa Vav was detected at 0.5 min and largely disappeared 5 min after CD19 cross-linking. When this blot was stripped and re-stained with anti-Vav, identical levels of Vav protein were present in all lanes (Fig. 4B). Therefore, the results in Fig. 4A cannot be ascribed to potential differences in the amount of immunoprecipitated Vav electrophoresed in each lane. In a separate experiment, 1E8 and BLIN-1 were cross-linked with anti-CD19, lysed, and subjected to rigorous pre-clearing by three rounds of immunoprecipitation with anti-CD19 or control IgG1. The lysates were then immunoprecipitated with anti-Vav and subjected to anti-p-Tyr immunoblotting. There was no detectable decrease in the intensity of the 95 kDa tyrosine phosphorylated Vav followed pre-clearing CD19, compared to pre-clearing with control IgG1 (data not shown). Thus, it is unlikely that the 95 kDa tyrosine phosphorylated protein in Fig. 4A is CD19.

Since Tuveson et al. reported that PI 3-kinase can bind to phosphorylated CD19 (7), we examined whether Vav interacts with the PI 3-kinase following CD19 cross-linking. Fig. 4C shows that the p85 subunit of PI 3-kinase was detected in the anti-Vav immunoprecipitates following 0.5 min of CD19 cross-linking in 1E8 and BLIN-1, whereas no PI 3-kinase was detected in anti-Vav immunoprecipitates from control IgG1 treated cells.

To more directly examine the potential interaction between Vav and PI 3-kinase, CD19 was cross-linked on 1E8 and BLIN-1 and cell lysates were immunoprecipitated with anti-PI 3-kinase (p85). As shown in Fig. 5A, anti-PI 3-kinase immunoprecipitates blotted with anti-p-Tyr revealed a 110 kDa molecule of unknown identity, which may be the catalytic subunit of PI 3-kinase. Control staining with anti-PI 3-kinase revealed that comparable amounts of immunoprecipitated protein were electrophoresed in each lane (Fig. 5B). Fig. 5C shows the same blot re-stained with anti-Vav, demonstrating that Vav was co-precipitated with PI 3-kinase in CD19 cross-linked cells. Although Vav was tyrosine phosphorylated following CD19 cross-linking (Fig. 4A), no tyrosine phosphorylation of co-precipitated Vav was detected (Fig. 5A). Tyrosine phosphorylation of the p85 subunit of PI 3-kinase has been observed following receptor-initiated signal transduction in lymphocytes (30,31). However, no tyrosine phosphorylation of p85 was detected following CD19 cross-linking (Fig. 5A).

Bustelo et al. reported that activation of the epidermal growth factor receptor or platelet-derived growth factor receptor leads to tyrosine phosphorylation of Vav, and Vav becomes physically coupled to the receptor through its SH2 domain (32). We therefore examined whether CD19 cross-linking induced an interaction between Vav and CD19. As shown in Fig. 6A, Vav was detected in the anti-CD19 immunoprecipitates following 1 min of CD19 cross-linking in 1E8 and BLIN-1, whereas anti-CD19 immunoprecipitates of control IgG1 treated cells only revealed weak staining of Vav. In a similar experiment, only weak staining of Vav (comparable to that in control IgG1 treated cells) was detected in the anti-CD19 immunoprecipitates of anti-class I MHC cross-linked 1E8 and BLIN-1 cells (data not shown). Interestingly, this increase in the Vav/CD19 interaction peaked at 1 min and decreased by 5 min, which correlated with the degree of tyrosine phosphorylation of Vav (Fig. 4A). There was no detectable Vav in control IgG1 immunoprecipitates of IgG1 treated cells. However, some Vav was detected in control IgG1 immunoprecipitates of anti-CD19 cross-linked cells. Since some CD19/Vav may complex to anti-CD19 used for cross-linking, it is likely that these immune complexes were precipitated by rabbit anti-mouse Ig-coated protein A-Sepharose beads during the immunoprecipitation procedure. The 185 and 105 kDa proteins present in the control IgG1 immunoprecipitates are non-specific proteins that were stained by secondary horseradish peroxidase-conjugated Ab in the ECL detection system. In Fig. 6B, the p85 subunit of PI 3-kinase was detected in anti-CD19 immunoprecipitates of CD19 cross-linked cells, whereas little PI 3-kinase was detected in control IgG1 treated cells, consistent with the report by Tuveson and colleagues (7). The appearance of some PI 3-kinase in the control IgG1 immunoprecipitates of CD19 cross-linked cells can probably be explained by rabbit anti-mouse precipitation of anti-CD19 immune complexes, as discussed above.

We then examined the effect of CD19 cross-linking on normal B cell precursors isolated from human fetal bone marrow. As shown in Fig. 9A, anti-p-Tyr immunoblotting revealed a modest increase in tyrosine phosphorylation of substrates of 55, 62, 95, 100, 110 and 125 kDa following CD19 cross-linking. When the blot was stripped and re-stained with anti-MAP kinase, no mobility retardation of p42 MAP kinase was detected following CD19 cross-linking (Fig. 9B). In contrast, phorbol 12-myristate 13-acetate (PMA) treatment resulted in faint tyrosine phosphorylation of a 43 kDa molecule in the anti-p-Tyr immunoblot (Fig. 9A), and induced pronounced mobility retardation of p42 MAP kinase (Fig. 9B). PMA treatment also induced mobility retardation of p42 MAP kinase in the pre-B cell line BLIN-1 (data not shown).

In order to more directly analyze activation of the MAP kinase pathway, we assayed for MEK activation following CD19 cross-linking using an in vitro kinase assay to assess the capacity of MEK to phosphorylate MAP kinase. Since MAP kinase is the only known substrate of MEK (35), a GST-MAP kinase fusion protein was used as a substrate in the in vitro kinase assay. As shown in Fig. 10, 1E8 and BLIN-1 cells treated with control IgG1 exhibited low MEK activity, based on the weakly phosphorylated GST-MAP kinase fusion protein. Phosphorylation of GST-MAP kinase was slightly enhanced by cross-linking class I MHC for 1 min, and more intensely enhanced by cross-linking CD19 for 1 or 5 min in both 1E8 and BLIN-1. Scanning densitometry was used to quantify the autoradiographic signals in Fig. 10. Anti-class I MHC cross-linking of 1E8 cells led to a 2.0-fold increase in GST-MAP kinase phosphorylation, and anti-CD19 cross-linking led to a 3.4-fold and 3.7-fold increase in GST-MAP kinase phosphorylation at 1 min and 5 min, respectively. Similarly, anti-class I MHC cross-linking of BLIN-1 cells led to a 1.8-fold increase in GST-MAP kinase phosphorylation, and anti-CD19 cross-linking led to a 2.4-fold and 3.4-fold increase in GST-MAP kinase phosphorylation at 1 min and 5 min, respectively.

The human B cell-specific cell surface molecule CD19 was identified and characterized over 10 years ago (1), and its complete amino acid sequence has been deduced from a cDNA (3). However, a putative CD19 ligand has not been described, and functional analyses have therefore relied on anti-CD19 mAb. Most of the functional studies on CD19 have focused on the relationship between CD19 and surface Ig signaling on B cells. Several reports have shown that anti-CD19 can inhibit the proliferation of B cells induced by anti-IgM (13-15). Other studies have demonstrated that anti-CD19 can enhance or inhibit, depending on the nature of the signaling event being modified or the identity of the responding cell (36,37). In contrast, using murine L cells transfected with the CD32 Fc receptor to present anti-CD19 or anti-IgM on the L cell surface, Carter et al. demonstrated that co-ligation of surface CD19 and Ig can reduce the Ig concentration required to induce the proliferation of mature B cells (16). Anti-CD19 presented on the L cells may mimic a putative cell bound CD19 ligand by delivering a potent cross-linking stimulus through CD19.

CD19 is expressed on B cell precursors at levels comparable to mature B cells (2,3). However, the function of CD19 on surface Ig negative B cell precursors has not been as thoroughly investigated. We have recently shown that IL-7 increases the level of cell surface CD19, and cross-linking CD19 triggers the homotypic aggregation of normal B cell precursors (18). We proposed (18) that anti-CD19 mAb 25C1 may activate a functional domain in a manner analogous to the putative CD19 ligand. We also found that the PTK inhibitor herbimycin A disrupted CD19-mediated homotypic aggregation in B cell precursors (W.-K. Weng and T.W. LeBien, unpublished observations), implicating PTK activation in CD19 signaling.

As an extension of our prior studies (18), we herein examined the activation of PTK following cross-linking of CD19. We observed PTK activation on both pre-B and immature B cell lines, as well as normal B cell precursors, which led to tyrosine phosphorylation of multiple electrophoretically distinct substrates in all cases (Figs. 2,7, and 9). Separate cross-linking of CD19 and IgM on the 1E8 immature B cell line activated PTK with similar kinetics, and revealed tyrosine phosphorylated proteins of similar sizes (Fig. 2). Comparable results have been reported using other B-lineage cells (38,39). This data suggests that PTK activated following cross-linking of CD19 and surface IgM may have common substrates. In this regard, CD19 undergoes tyrosine phosphorylation following cross-linking of both surface IgM and CD19 (7,38,39). We have now identified one of the tyrosine phosphorylated substrates in CD19 cross-linked cells as Vav, which has recently been demonstrated to undergo rapid and transient tyrosine phosphorylation following B cell antigen receptor-activated signal transduction (22). In both pre-B and immature B cell lines Vav was tyrosine phosphorylated within 1 min after CD19 cross-linking (Fig. 4A). However, the amount of Vav detected in anti-p-Tyr immunoprecipitates remained steady or slightly increased between 1 and 5 min after CD19 cross-linking (Fig.3), whereas tyrosine phosphorylation of Vav substantially decreased between 1 and 5 min after CD19 cross-linking (Fig. 4A). This suggests that in addition to the tyrosine phosphorylation of Vav, Vav may also associate with tyrosine phosphorylated proteins (which could include other proteins in addition to CD19) following CD19 cross-linking. Our results establish a potentially important link between CD19 cross-linking and tyrosine phosphorylation of Vav, which has implications for activation of the Ras pathway (see below). It is also possible that a Ras-independent pathway may be activated through Vav. This possibility receives support from the knowledge that Vav has multiple (potential) functional domains, including an amino-terminal helix-loop-helix/leucine zipper motif (40,41) and two nuclear localization signals (42).

One of the most important observations in the current study was the discovery of a potentially complex physical interaction between CD19, Vav, and the p85 subunit of PI 3-kinase. Cross-linking CD19 leads to the association of tyrosine phosphorylated CD19 with p85 via two Tyr-X-X-Met motifs in the CD19 cytoplasmic tail (7). This interaction serves to activate the p110 catalytic subunit of PI 3-kinase (7), although the mechanism of activation is unknown. The CD19/p85 association was also predicted based on the use of phosphopeptide libraries to determine the peptide-binding site specificity of SH2 domains (5). Our data in Fig. 6B are consistent with a CD19/p85 physical association enhanced by CD19 cross-linking. This CD19/p85 interaction depends on the tyrosine phosphorylation of CD19 (7), therefore, it can also be used as an indirect indication of tyrosine phosphorylation of CD19. Since the CD19/p85 interaction was detected after 1 min CD19 cross-linking and started to decrease by 10 min, this suggests that CD19 was tyrosine-phosphorylated between 1 and 10 min following cross-linking. Furthermore, the collective data in Figs. 4 and 5 provide strong evidence that CD19 cross-linking induces an increase in the physical interaction between p85 and Vav. This activation induced interaction between p85 and Vav has, to our knowledge, not been described. Our studies did not delineate the mechanism by which Vav and p85 interact. However, the proline rich region of the p85 subunit can interact with SH3 domains of Fyn and Lyn, and this interaction increases the specific activity of PI 3-kinase (43). It remains to be determined whether either of the two SH3 domains in Vav can interact with the proline rich region in p85. Interaction via the Vav SH2 domain seems unlikely, since no detectable tyrosine phosphorylation of p85 occurred following CD19 cross-linking (Fig. 5A). Whereas CD19 cross-linking induced the tyrosine phosphorylation of Vav (Fig. 4A), no tyrosine phosphorylation of p85-associated Vav was detected (Fig. 5A). There are two possible explanations for the latter result. First, only non-phosphorylated Vav may associate with p85 following CD19 cross-linking. Second, only a fraction of p85-associated Vav may become tyrosine phosphorylated, which may be difficult to detect by anti-p-Tyr immunoblotting.

In addition to the p85/CD19 and p85/Vav interactions, we also observed that CD19 cross-linking enhanced the interaction between Vav and CD19 (Fig. 6). This interaction could be mediated through the SH2 domain of Vav, since a Tyr-Glu-Glu-Pro motif in CD19 has been proposed to specifically bind to the Vav SH2 domain (6). It is also possible that the intereaction between Vav and CD19 is an indirect one via a third protein, e.g. the p85 subunit of PI 3-kinase. Lyn and Lck Src-family PTK have been reported to associate with CD19, and their enzymatic activities increase following CD19 cross-linking (38,44). This enhanced interaction between CD19 and Vav may facilitate the physical translocation of Vav into close proximity to activated PTK, thereby stimulating the tyrosine phosphorylation and subsequent activation of Vav GTP/GDP exchange activity. However, it is not known whether Lyn, Lck, or other Src-family PTK phosphorylate Vav.

In conclusion, we have demonstrated that surface Ig negative and surface Ig positive human B-lineage cells respond to CD19 cross-linking by PTK activation and formation of a physical complex (of unknown stoichiometry) consisting of CD19, Vav, and PI 3-kinase. One interpretation of this data would hold that two distinct signaling pathways are activated through CD19, one involving Vav/Ras/MAP kinase, and the other involving PI 3-kinase. Future studies will need to determine the changes in gene expression that occur in B-lineage cells as a consequence of these pathways being activated, and the ultimate effect of these events on the survival, growth, and differentiation of early B-lineage cells.

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We thank Dr. Julia G. Johnson for her technical assistance and valuable discussions; Dr. Frederick T. Boyd for his help with the densitometric quantitation; and Nisha Shah and Julie Rehmann Pribyl for technical assistance. We also thank Dr. Robert Abraham, Mayo Clinic, for his very helpful comments, and Dr. Che-Leung Law, University of Washington School of Medicine, for his critical review of the manuscript.

* This work was supported by Grants R01 CA-31685 from the National Cancer Institute/National Institutes of Health and the Leukemia Task Force. W.-K. Weng is the recipient of a predoctoral fellowship from NIH Immunology Training Grant T32 AI-07313.

‡ Address correspondence and reprint requests to Tucker W. LeBien, PhD, Box 609 UMHC, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, 420 Delaware St. SE, Minneapolis, MN 55455

1 Abbreviations used in this paper: Ig, immunoglobulin; PTK, protein tyrosine kinase; PI 3-kinase, phosphatidylinositol 3-kinase; anti-p-Tyr, anti-phosphotyrosine mAb; MAP kinase, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase and extracellular signal-regulated kinase kinase; mAb, monoclonal antibody; MHC, major histocompatability complex; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate.