Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton's tyrosine kinase

Bruton's tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD19 mutant in which tyrosine residues previously shown to mediate CD19 interaction with PI3-K, Y484 and Y515, were changed to phenylalanine. Taken together, the data presented indicate that BCR aggregation-driven CD19 phosphorylation functions to promote Btk activation via recruitment and activation of PI3-K. Resultant phosphatidylinositol 3,4,5-trisphosphate probably functions to localize Btk for subsequent phosphorylation and activation by Src and Syk family kinases.     

Differential regulation of the B cell receptor-mediated signaling by the E3 ubiquitin ligase Cbl

The E3 ubiquitin ligase Cbl has been implicated in intracellular signaling pathways induced by the engagement of the B cell antigen receptor (BCR) as a negative regulator. Here we showed that Cbl deficiency results in a reduction of B cell proliferation. Cbl-/- B cells show impaired tyrosine phosphorylation, reduced Erk activation, and attenuated calcium mobilization in response to BCR engagement. The phosphorylation of Syk and Btk is also down-modulated. Interestingly, Cbl-/- B cells display enhanced BCR-induced phosphorylation of CD19 and its association with phosphatidylinositol 3-kinase. Importantly, Lyn kinase activity is up-regulated in Cbl-/- B cells, which correlates inversely with the Cbl-mediated ubiquitination of Lyn. Because Lyn has both negative and positive roles in B cells, our results suggested that Cbl differentially modulates the BCR-mediated signaling pathways through targeting Lyn ubiquitination, which affects B cell development and activation.     

B cell antigen receptor-induced activation of Akt promotes B cell survival and is dependent on Syk kinase

Signaling through the B cell Ag receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not known. Using the chicken B lymphoma cell line DT40 as a model system, we investigated the role of the serine/threonine kinase Akt in B cell activation. While parental DT40 cells undergo apoptosis in response to BCR cross-linking, cells overexpressing Akt show a greatly diminished apoptotic response. By contrast, limiting the activation of Akt, either by inhibiting phosphatidylinositol 3-kinase or by ectopic expression of the phospholipid phosphatase MMAC1, results in a significant increase in the percentage of apoptotic cells after BCR cross-linking. Using various DT40 knockout cell lines, we further demonstrate that the tyrosine kinase Syk is required for Akt activation and that Lyn tyrosine kinase inhibits Akt activation. Taken together, the data demonstrate that Akt plays an important role in B cell survival and that Akt is activated in a Syk-dependent pathway.     

BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP(3) receptor.

B-cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. Here we report on the identification and characterization of the B-cell scaffold protein with ankyrin repeats (BANK), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation, which is mediated predominantly by Syk. Overexpres sion of BANK in B cells leads to enhancement of BCR-induced calcium mobilization. We found that both Lyn and inositol 1,4,5-trisphosphate receptor (IP(3)R) associate with the distinct regions of BANK and that BANK promotes Lyn-mediated tyrosine phosphorylation of IP(3)R. Given that IP(3)R channel activity is up-regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR-induced calcium mobilization by connecting PTKs to IP(3)R. Because BANK expression is confined to functional BCR-expressing B cells, BANK-mediated calcium mobilization may be specific to foreign antigen-induced immune responses rather than to signaling required for B-cell development.     

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.     

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.     

Cross-linking of the B cell receptor induces activation of phospholipase D through Syk, Btk and phospholipase C-gamma2

Phospholipase D (PLD) has been proposed to play a key role in the signal transduction of cellular responses to various extracellular signals. Herein we provide biochemical and genetic evidence that cross-linking of the B cell receptor (BCR) induces rapid activation of PLD through a Syk-, Btk- and phospholipase C (PLC)-gamma2-dependent pathway in DT40 cells. Activation of PLD upon BCR engagement is completely blocked in Syk- or Btk-deficient cells, but unaffected in Lyn-deficient cells. Furthermore, in PLC-gamma2-deficient cells, BCR engagement failed to activate PLD. These results demonstrate that Syk, Btk and PLC-gamma2 are essential for BCR-induced PLD activation.     

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.     

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.     

Latent membrane protein 2A-mediated effects on the phosphatidylinositol 3-Kinase/Akt pathway

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membranes of B lymphocytes and blocks B-cell receptor (BCR) signaling in EBV-transformed B lymphocytes in vitro. The phosphotyrosine motifs at positions 74 or 85 and 112 within the LMP2A amino-terminal domain are essential for the LMP2A-mediated block of B-cell signal transduction. In vivo studies indicate that LMP2A allows B-cell survival in the absence of normal BCR signals. A possible role for Akt in the LMP2A-mediated B-cell survival was investigated. The protein kinase Akt is a crucial regulator of cell survival and is activated within B lymphocytes upon BCR cross-linking. LMP2A expression resulted in the constitutive phosphorylation of Akt, and this LMP2A effect is dependent on phosphatidylinositol 3-kinase activity. In addition, recruitment of Syk and Lyn protein tyrosine kinases (PTKs) to tyrosines 74 or 85 and 112, respectively, are critical for LMP2A-mediated Akt phosphorylation. However, the ability of LMP2A to mediate a survival phenotype downstream of Akt could not be detected in EBV-negative Akata cells. This would indicate that LMP2A is not responsible for EBV-dependent Burkitt's lymphoma cell survival.     

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLCgamma2 and to maximize its catalytic efficiency for IP3 production.     

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.     

Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling

Activation of phospholipase C-gamma2 (PLCgamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCgamma2.     

Src family kinases mediate receptor-stimulated, phosphoinositide 3-kinase-dependent, tyrosine phosphorylation of dual adaptor for phosphotyrosine and 3-phosphoinositides-1 in endothelial and B cell lines

DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor beta (PDGFbeta) receptors, which express Bmx and Src as their major Btk (Bruton's tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFbeta (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated approximately 10-fold by PtdIns(3,4,5)P(3) (IC(50) = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P(3)-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane.     

Early signaling events induced by 280-nm UV irradiation.

The depletion of stratospheric ozone results in increased UV (ultraviolet) light below 300 nm, and has significant effects on biological systems. To better understand the effects of UV in this range, early signaling events induced by monochromatic UV light were investigated using the chicken B cell line DT40 and mutants lacking protein tyrosine kinases (PTKs). Among MAP kinase family proteins, P38 MAP kinase (P38) was selectively and immediately activated by 280 nm UV light in cultured DT40 cells. Activation of P38 was completely inhibited in cells deficient in Lyn and Btk. Introduction of wild-type Btk, but not kinase-inactive Btk, restored the P38 activation. In contrast, P38 activation was not affected in Syk-deficient cells. Tyrosine phosphorylation of Lyn was induced by 280 nm UV light, and pretreatment of cells with orthovanadate, an inhibitor of protein tyrosine phosphatase (PTP), enhanced both Lyn phosphorylation and P38 activation. These results show that Lyn and Btk are upstream regulators of the P38 signaling pathway activated by 280 nm UV light and that the triggering event likely involves inactivation of PTP. Furthermore, cell death induced by 280 nm UV irradiation were augmented by Btk depletion or a specific inhibitor for P38, and partially blocked in Lyn-deficient cells, suggesting that the Lyn-Btk-P38 pathway promotes cell survival while other Lyn pathways stimulate cell death.     

Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

The Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of lambda L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of lambda L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted lambda usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-kappaB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of lambda L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity.     

The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32) regulates B cell antigen receptor internalization

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32-/- and Lyn-/- cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32-/- cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.