Deregulated Syk inhibits differentiation and induces growth factor–independent proliferation of pre–B cells

The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor–independent proliferation and transforms bone marrow–derived pre–B cells that are then able to induce leukemia in mice. Syk-transformed pre–B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre–B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre–B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc–transformed pre–B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre–B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes

BCL6 is critical for the development of a diverse primary B cell repertoire

BCL6 protects germinal center (GC) B cells against DNA damage–induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7–dependent B cell precursors, we report that IL-7Rα–Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a μ heavy chain, however, activation of pre–B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Rα–Stat5 signaling is attenuated. At the transition from IL-7–dependent to –independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre–B cells from DNA damage–induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre–B cell self-renewal and the formation of a diverse polyclonal B cell repertoire

B cell antigen receptor assembly and Syk activation in the S2 cell reconstitution system

Signal transduction from the B cell antigen receptor (BCR) involves a multitude of signaling molecules often organized in dynamic protein complexes. The molecular mechanisms operating during signaling are difficult to study solely by loss-of-function analysis. For a better understanding of the transient interaction of signaling molecules and their regulation by feedback loops, as well as their dynamic behavior in living cells, new techniques are required. We have developed a method allowing the reconstitution of the BCR complex and several of its key signaling elements in the evolutionary distant environment of the Drosophila S2 Schneider cell line. With this gain-of-function approach, we study here the assembly of the BCR complex and the control of its transport to the cell surface of S2 cells. We find that without binding to a light chain, the membrane-bound μm heavy chain (μmHC) homodimer, together with the Ig-α/Ig-β heterodimer, can come to the cell surface where it is signaling competent. This finding could have implications for potential signaling functions of such a receptor molecule during pro-/pre-B cell development. We also studied the activation of the BCR-proximal kinase Syk. We found that a truncated Syk mutant lacking the first (N-terminal) SH2 domain and the linker regions, is still regulated by autoinhibition and can only become activated in the presence of the BCR. This indicates that the C-terminal SH2 domain of Syk is the dominant regulatory subunit of this kinase

Conventional Light Chains Inhibit the Autonomous Signaling Capacity of the B Cell Receptor

Signals from the B cell antigen receptor (BCR), consisting of μ heavy chain (μHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of μHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca2+ flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of μHC to induce Ca2+ flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous μHC ability and resulted in antigen-dependent Ca2+ flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation

Amplification of B Cell Antigen Receptor Signaling by a Syk/ITAM Positive Feedback Loop

We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive llosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H₂O₂ and the redox equilibrium inside the cell

Identification of a Pre-BCR Lacking Surrogate Light Chain

SLP-65-/- pre-B cells show a high proliferation rate in vitro. We have shown previously that λ5 expression and consequently a conventional pre-B cell receptor (pre-BCR) are essential for this proliferation. Here, we show that pre-B cells express a novel receptor complex that contains a μ heavy chain (μHC) but lacks any surrogate (SL) or conventional light chain (LC). This SL-deficient pre-BCR (SL-pre-BCR) requires Ig-α for expression on the cell surface. Anti-μ treatment of pre-B cells expressing the SL-pre-BCR induces tyrosine phosphorylation of substrate proteins and a strong calcium (Ca2+) release. Further, the expression of the SL2+-pre-BCR is associated with a high differentiation rate toward κLC-positive cells. Given that B cell development is only partially blocked and allelic exclusion is unaffected in SL-deficient mice, we propose that the SL-pre-BCR is involved in these processes and therefore shares important functions with the conventional pre-BCR

N-linked glycosylation selectively regulates autonomous precursor BCR function

Developing B cells express distinct classes of B cell antigen receptors (BCRs) that differ in their heavy chain (HC). Although only μHC is expressed in early stages, δHC-containing BCRs dominate on the surface of mature B cells. The reason for the tightly regulated expression of these receptors is poorly understood. Here we show that μHC was specifically required for precursor BCR (pre-BCR) function and that δHC was unable to form a functional pre-BCR. A conserved asparagine (N)-linked glycosylation site at position 46 (N46) in the first conserved domain of μHC was absolutely required for pre-BCR function, and swapping that domain with δHC resulted in a functional δHC-containing pre-BCR. When tested in the context of the BCR, μHC with a mutant N46 showed normal function, which indicated that N46-glycosylation is specifically required for pre-BCR function. Our results suggest an unexpected mode of pre-BCR function, in which binding of the surrogate light chain to N46 mediates autonomous crosslinking and, concomitantly, receptor formation