Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase C.

Abstract

It has been found that the principal biochemical pathway activated in B cells stimulated by antigen-or anti-immunoglobulin-mediated crosslinking of surface immunoglobulin is that resulting in hydrolysis of phosphatidylinositol bisphosphate with generation ofdiacylglycerol and inositol trisphosphate. Recent evidence suggests that surface immunoglobulin-mediated B-cell activation can proceed without detectable increases in the concentration of either diacylglycerol or intracellular Ca2+ concentration, implicating involvement of other non-protein-kinase-C/Ca2+-dependent signal-transduction pathways. Therefore, we sought evidence for activation of a signalin pathway that is associated with growth regulation in other cell types-i.e., the protein-tyrosine kinases. We now show that crosslinking of membrane immunoglobulin by mitogenic antibodies leads to rapid tyrosine phosphorylation of several cellular substrates, consistent with the induction of a tyrosine kinase activity. This increase in tyrosine phosphorylation is weakly (if at all) stimulated by other B-cell mitogens, including phorbol esters and ionophores, and does not require the presence of detectable protein kinase C. Furthermore, inhibition ofanti-immunglobulin-stimulated phosphatidylinositol bisphosphate hydrolysis does not inhibit activation of this tyrosine kinase-dependent pathway. These findings suggest that occupancy of the membrane immunglobulin receptor may induce multiple pathways of activation.Antigen interaction with the B-cell surface immunoglobulin (sIg) receptor stimulates resting B cells to proliferate and, in the presence of T cells or T cell-derived lymphokines, to secrete immunoglobulin (1)(2)(3)(4). While the biochemical mechanisms that underlie B-cell activation are beginning to be unraveled, there is one pathway of activation that has received considerable attention. Many investigators have demonstrated that crosslinking of sIg leads to hydrolysis of phosphatidylinositol bisphosphate (Ptdlns-P2) with the generation of diacylglycerol and inositol trisphosphate (InsP3) (5-9). The released diacylglycerol activates protein kinase C (PKC) (10), and inositol trisphosphate leads to a rise in intracellular calcium ([Ca2+Ji) (11). This pathway was thought to be the major, if not only, sIg-mediated signaling pathway in B cells. We and others have demonstrated that antiimmunoglobulin (anti-Ig)-mediated B-cell stimulation can proceed in the absence of cellular PKC, implicating a role for a non-PtdIns-P2-dependent pathway of sIg activation (12, 13). More recently, we have shown that dextran-conjugated anti-Ig at pg/ml concentrations stimulates B-cell proliferation in the absence ofdetectable PtdIns-P2 hydrolysis or increases in [Ca2+], (14,15), thus also raising the possibility that other PtdIns-P2-independent pathways are being recruited during sIg-mediated signaling. A third line of evidence supporting this possibility was our recent finding that inhibition of anti-Ig-stimulated PtdIns-P2 hydrolysis by PKC activators enhanced rather than...

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

“…Next, we examined the changes in tyrosine phosphorylation of cellular proteins after BCR stimulation in wild-type and Csk-negative cells. As observed with other B cell lines (5,11,18), wild-type DT40 cells showed only a marginal level of protein tyrosine phosphorylation without BCR stimulation (Fig. 3).…”

“…One feature in the regulation of Src PTKs in lymphoid cells different from that in fibroblasts is that tyrosine phosphorylation of Src PTKs is barely detectable unless cells are stimulated by a variety of ligands, such as those against the TCR and BCR, as we have observed in DT40 cells (5,11,18,47). In fibroblasts, it is well-known that c-Src is predominantly phosphorylated at the C-terminal tyrosine residue (Tyr-527), suppressing the kinase activity (28).…”

“…In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that (48). Experimentally, this process can be mimicked by cross-linking of the B-cell antigen receptor (BCR) with anti-BCR antibodies to induce a rapid elevation of protein tyrosine phosphorylation, an increase in inositol 1,4,5-trisphosphate (1P3), and an elevation of the intracellular calcium concentration (4,5,11,12,18,32,48).The BCR is composed of multiple subunits, the antigen recognition components Iga (MB-1) and Igf (B29) (22, 23). The BCR subunits do not possess an intrinsic tyrosine kinase activity; however, the BCR has been shown to be coupled functionally with at least two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Src PTKs) and Syk (7,10,44,49).…”

“…The stimulation of these receptors triggers a cascade of intracellular events (48). Experimentally, this process can be mimicked by cross-linking of the B-cell antigen receptor (BCR) with anti-BCR antibodies to induce a rapid elevation of protein tyrosine phosphorylation, an increase in inositol 1,4,5-trisphosphate (1P3), and an elevation of the intracellular calcium concentration (4,5,11,12,18,32,48).…”

Induction of bcl-2 Expression by Phosphorylated CREB Proteins during B-Cell Activation and Rescue from Apoptosis

“…Binding of antigen to the B-cell receptor in resting B cells leads to proliferation. Binding of antigen to the B-cell receptor on either germinal-center cells or resting B cells leads to a similar pattern of second-messenger induction, including activation of Src and Src-related protein kinases (4,60), phosphorylation on tyrosine residues of a number of substrates (3,6,20,31), engagement of G proteins (19,25,39), increased inositol 1,4,5-triphosphate levels, increased intracellular calcium levels, and activation of protein kinase C (PKC) (5,9). Activation of PKC leads to activation of NF-B and AP-1, as well as other transcription factors (10,34,54).…”