Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis.

Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.

c-Cbl negatively regulates platelet activation by glycoprotein VI.

BACKGROUND: The adapter protein c-Cbl has emerged as having a potential role in negative regulation of immune receptor signaling. The major platelet-signaling receptor for collagen, glycoprotein VI (GpVI), is associated with the Fc receptor (FcR) gamma-chain, and signals through a similar pathway to immune receptors. c-Cbl is tyrosine-phosphorylated in response to stimulation of GpVI, whereas phosphorylation of c-Cbl in thrombin-activated platelets is dependent on fibrinogen binding to the integrin GpIIb/IIIa. OBJECTIVE: To investigate the role of c-Cbl in platelet signaling. METHODS: Murine platelets lacking functional c-Cbl or Src family kinases were analyzed. RESULTS: Phosphorylation of c-Cbl through GpVI is reduced in murine platelets deficient in the Src-family kinases Fyn and Lyn, demonstrating that they lie upstream of c-Cbl phosphorylation. Phosphorylation of several proteins of the GpVI-signaling pathway, including the FcR gamma-chain, Syk and phospholipase Cgamma2 (PLCgamma2), is increased in the absence of c-Cbl. In line with this, aggregation is potentiated in response to the GpVI-specific collagen-related peptide (CRP) after a slight delay. A delay in potentiation is also seen in response to stimulation by thrombin. CONCLUSIONS: These observations demonstrate that c-Cbl negatively regulates platelet responses to GpVI agonists and to thrombin, with the latter effect possibly being mediated downstream of GpIIb/IIIa. c-Cbl may play a physiological role in helping to prevent unwanted platelet activation in vivo.

Regulation of phospholipase C gamma isoforms in haematopoietic cells: why one, not the other?

Phospholipase C gamma (PLCgamma) isoforms are critical for the generation of calcium signals in haematopoietic systems in response to the stimulation of immune receptors. PLCgamma is unique amongst phospholipases in that it is tightly regulated by the action of a number of tyrosine kinases. It is itself directly phosphorylated on a number of tyrosines and contains several domains through which it can interact with other signalling proteins and lipid products such as phosphatidylinositol 3,4,5-trisphosphate. Through this network of interactions, PLCgamma is activated and recruited to its substrate, phosphatidylinositol 4,5-bisphosphate, at the membrane. Both isoforms of PLCgamma, PLCgamma1 and PLCgamma2, are present in haematopoietic cells. The signalling cascade involved in the regulation of these two isoforms varies between cells, though the systems are similar for both PLCgamma1 and PLCgamma2. We will compare these cascades for both PLCgamma1 and PLCgamma2 and discuss possible reasons as to why one form of PLCgamma and not the other is required for signalling in specific haematopoietic cells, including T lymphocytes, B lymphocytes, platelets, and mast cells.

Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation.

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

ADP-induced platelet shape change: an investigation of the signalling pathways involved and their dependence on the method of platelet preparation.

Shape change is an important early event in platelet activation. In this study we show that the Ca2+ chelator BAPTA and the Rho-kinase inhibitor Y-27632 inhibit ADP-induced myosin light chain (MLC) phosphorylation and platelet shape change through distinct pathways and with distinct kinetics. Ca2+ is largely responsible for the initial onset of shape change, whilst Rho-kinase plays a major role in the maintenance of the response. The relative contribution of these two pathways to each stage of the response was dependent on the method of platelet preparation, but in all cases shape change was shown to be downstream of the P2Y1 receptor. Similar observations were made in murine platelets. The shape change response was modulated via changes in cAMP levels, possibly via the P2TAC receptor, but not by tyrosine phosphorylation. We conclude that ADP-induced shape change occurs via the P2Y1 receptor, which can be differentially coupled to Rho-kinase and Ca2+-linked pathways dependent on the method of platelet preparation.

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.

Up-regulation of p21- and RhoA-activated protein kinases in human pregnant myometrium.

The role of small ras homologous GTP-binding proteins in the regulation of smooth muscle contractility has become increasingly apparent but there is still little information about the presence of these proteins in human uterine smooth muscle. Messenger RNAs for p21-activated protein kinase isoforms (PAK1, PAK2, and PAK3) were detectable in both nonpregnant and pregnant human myometrial tissue. However, PAK3 protein was not detectable and the proteins for PAK1 and PAK2 were only detectable in pregnant tissue. Moreover there was a large increase in the constitutively active p34 protein fragment of PAK2 in pregnant tissue. Protein expression of RhoA-activated protein kinases isoforms (ROK1 and ROK2) also increased during pregnancy. Stimulation of RhoA signaling in pregnant myometrial tissue with lysophosphatic acid (LPA) increased the level of myosin light chain (MLC20) phosphorylation. Preincubation of the tissue with C3 toxin inhibited LPA-stimulated MLC20 phosphorylation and lowered the basal phosphorylation level of MLC20. Thus ROKS and PAKS have the potential to regulate uterine contractility and/or load-bearing during human pregnancy.

Regulation and function of WASp in platelets by the collagen receptor, glycoprotein VI.

Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin alpha2beta1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.

The ordered visual transduction complex of the squid photoreceptor membrane.

The study of visual transduction has given invaluable insight into the mechanisms of signal transduction by heptahelical receptors that act via guanine nucleotide binding proteins (G-proteins). However, the cyclic-GMP second messenger system seen in vertebrate photoreceptor cells is not widely used in other cell types. In contrast, the retina of higher invertebrates, such as squid, offers an equally accessible transduction system, which uses the widespread second messenger chemistry of an increase in cytosolic calcium caused by the production of inositol-(1,4,5)-trisphosphate (InsP3) by the enzyme phospholipase C, and which may be a model for store-operated calcium influx. In this article, we highlight some key aspects of invertebrate visual transduction as elucidated from the combination of biochemical techniques applied to cephalopods, genetic techniques applied to flies, and electrophysiology applied to the horseshoe crab. We discuss the importance and applicability of ideas drawn from these model systems to the understanding of some general processes in signal transduction, such as the integration of the cytoskeleton into the signal transduction process and the possible modes of regulation of store-operated calcium influx.

Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets.

Both Rho-kinase and the Ca(2+)/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca(2+). Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca(2+) and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein-coupled platelet receptors, independent of an increase in cytosolic Ca(2+). Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca(2+)/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).