Che-Leung Law

Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cγ2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor γ-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor γ-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor γ-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin α2β1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcγRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor γ-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin α2β1 and involves phosphorylation of the Fc receptor γ-chain, its association with Syk and subsequent phosphorylation of phospholipase Cγ2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.

Protein Kinase C μ (PKCμ) Associates with the B Cell Antigen Receptor Complex and Regulates Lymphocyte Signaling

We have identified a Ser/Thr kinase associated with the B cell receptor (BCR) complex as protein kinase C mu (PKC mu). PKC mu activity is up-regulated after cross-linking the BCR and CD19 on B cells, and PKC mu co-precipitates with Syk and phospholipase C-gamma 1/2 (PLC gamma 1/2). In vitro phosphorylation of fusion proteins showed that both Syk and PLC gamma 1 are potential substrates of PKC mu in vivo. Analysis of mutants of the chicken B cell line DT40 deficient in either Syk, Lyn, Btk, or PLC gamma 2 revealed that BCR-induced activation of PKC mu, like activation of PLC gamma 2, requires Syk and is partially regulated by Btk, but is Lyn independent. PKC mu can down-regulate the ability of Syk to phosphorylate PLC gamma 1 in vitro. Thus, PKC mu may function in a negative feedback loop regulating BCR-initiated signaling cascades.

Regulation of lymphocyte activation by the cell-surface molecule CD22

Accessory molecules play an important role in the regulation of lymphocyte activation mediated by the B-cell antigen receptor (BCR). CD22 is one such accessory molecule expressed on B-lineage cells. Here, Che-Leung Law and colleagues review current knowledge on the structure-function relationship between CD22 and the BCR, discuss the role of CD22 as a cell-adhesion molecule and suggest models for potential in vivo functions of CD22.

Regulation of signalling through B-lymphocyte antigen receptors by cell-cell interaction molecules

Expression of clonotypic antigen receptors (AgR) on lymphocytes is required for vertebrates to combat foreign antigens. Since somatic rearrangement ofthe receptor genes and hypermutations are random processes, the immune system has evolved mechanisms to ensure elimination oflymphocytes expressing self-reactive AgR while allowing the functional development of lymphocytes directed against foreign antigens. In the B-Iineage, structurally related pre-B-cell receptor and mature B-cell receptor (BCR) complexes are expressed on B-cell precursors and membrane Ig^ (mig) B cells, respectively. Although the pre-B-cell receptor and the BCR share similar structural features, signals transduced by them elicit distinct responses. In pre-B cells, a proliferative signal is presumably delivered by pre-B-cell receptors. B-cell precursors with non-productive receptor gene rearrangements and no functional pre-B-cell receptors may die because of their failure to receive a positive signal (death by neglect) (Melchers et al. 1995). In contrast, ligation of the BCR on immature IgM^/IgD B cells emerging from the bone marrow delivers an apoptotic signal to these cells leading to activationinduced eel] death, a mechanism believed to be responsible for the elimination of self-reactive B cells (Melchers et al. 1995). As mature B cells are being selected for their ability to produce high-affinity antibodies in lymph node germinal centers (GCs), signals transduced by the BCR become positive once again (MacLennan 1994). Thus, any BCR can mediate both positive and negative sig-

Cell-cell interactions that regulate the development of B-lineage cells

Significant progress has been made recently in our understanding of the functions of lymphocyte-associated surface proteins. The latest developments involve the identification of ligands or co-receptors for many of these surface proteins. The signal transduction mechanisms utilized by these molecules are also beginning to be elucidated.

FcγRII tyrosine phosphorylation differs between FcγRII cross-linking and platelet-activating anti-platelet monoclonal antibodies

Abstract Using glutathione S -transferase Syk fusion proteins, we evaluated the mode of platelet FcγRII tyrosine phosphorylation induced by FcγRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcγRII with FcγRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcγRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcγRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcγRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcγRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcγRII-dependent platelet activation without FcγRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcγRII cross-linking.