Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation

Abstract

Platelet activation by newly exposed basement membrane collagen is a key initial step in both hemostasis and thrombosis (1). The complex of glycoprotein VI (GPVI) 2 and the Fc receptor ␥-chain is primarily responsible for activation of platelets by collagen (2). This receptor acts via a PLC␥2-dependent pathway, leading to generation of autocoids that recruit additional platelets to the site of injury. Signaling downstream of the GPVI/Fc receptor ␥ chain is similar to signaling initiated by activation of immune receptors on T-and B-lymphocytes. The Fc receptor ␥-chain contains an immunoreceptor tyrosine activation motif. Binding of collagen to the receptor is thought to cluster the receptors, which initiates the phosphorylation of the immunoreceptor tyrosine activation motif tyrosines by an Src family kinase. Similar to signaling through other immune receptors, Syk tyrosine kinase binds to the phosphorylated immunoreceptor tyrosine activation motif to start a cascade of phosphorylations and protein interactions resulting in the activation of PLC␥2.Hematopoietic cells express two members of the Cbl family of E3 ligases namely, c-Cbl and Cbl-b (3-5). The Cbl family proteins have been shown to be key regulators of intracellular signaling in immune cells (6, 7). As E3 ligases, Cbl proteins can catalyze the transfer of ubiquitin molecules to their substrates. A primary role for ubiquitylation is targeting the substrates of E3 ligases to the proteasome, where the substrates are proteolytically degraded. Because of this activity, Cbl family members have come to be considered as negative regulators of signaling. In platelets, c-Cbl promotes ubiquitylation of Syk and appears to be a negative regulator of platelet activation. Murine platelets deficient in c-Cbl show enhanced platelet aggregation in response to convulxin, a GPVI agonist (8, 9). Furthermore, we have shown that Syk is not ubiquitylated in c-Cbl Ϫ/Ϫ platelets, in contrast to normal platelets (9).The presence of Cbl-b in platelets and any role it might have in signaling have not been investigated. Because Cbl-b is also an E3 ligase, one might expect that it would also be a negative regulator of cell function. However, several reports have indicated that Cbl-b may play a positive role in both T cell and B cell signaling. Cbl-b interacts with Zap-70, leading to activation of T-cell-specific transcription factor NF-AT (10). Cbl-b Ϫ/Ϫ DT40 B cells display reduced PLC␥2 activation and Ca 2ϩ mobilization upon B-cell receptor stimulation, and the overexpression of Cbl-b results in an enhanced agonist-dependent Ca 2ϩ mobilization (11). These results may be explained by another function of the Cbl molecules, namely that they have several domains in addition to those responsible for the E3 ligase activity, which, acting as a scaffold, can bind to a variety of signaling molecules, bringing them into close proximity (6,12,13).Given the importance of Cbl-b to regulation of signaling in lymphocytes, we have investigated whether Cbl-b regulates platelet activation downstream ...

Activation of phosphatidylinositol 3-kinase β by the platelet collagen receptors integrin α2β1 and GPVI: The role of Pyk2 and c-Cbl

“…Although we confirmed the hyperphosphorylation of Syk in c-Cbl deficient platelets, phosphorylation of Akt was not significantly increased, indicating that c-Cbl does not target the PI3K pathway to limit platelet activation by GPVI. Platelets also express Cbl-b, a second member of the Cbl family, that, in differently to c-Cbl, has been implicated in GPVI-mediated platelets activation [32]. The antibody used in the present study is specific for c-Cbl as indicated in the data sheet, and thus whether or not Cbl-b may contribute to PI3Kβ activation and Akt phosphorylation by GPVI or by integrin α2β1 remains to be established.…”

“…Activated growth factor receptors, cytosolic tyrosine kinases, as well as phosphorylated adaptor proteins can interact with p85 and cause the activation of p110 catalytic subunit [14]. Among these, the adaptor protein c-Cbl is a known binding partner and activator of PI3K [29][30][31][32][33][34], and has been shown to associate with PI3K in integrin αIIbβ3 outside-in signaling, and to be tyrosine phosphorylated by Pyk2 (15). Here we demonstrated that upon integrin α2β1 engagement c-Cbl interacts with PI3K but is not tyrosine-phosphorylated.…”

RhoG Protein Regulates Glycoprotein VI-Fc Receptor γ-Chain Complex-mediated Platelet Activation and Thrombus Formation

“…Flow Cytometry-Washed murine platelets were used to measure the agonist-dependent level of activated ␣IIb␤3 receptors with phycoerythrin-conjugated antibody JON/A and the surface exposure of P-selectin with FITC-conjugated anti-Pselectin antibody as described previously (29). All determinations were performed on a FACSCalibur flow cytometer (BD Biosciences).…”

Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling

“…After protein determination (BCA protein assay kit, Thermo Scientific), 40-80 g of total cell lysate was electrophoresed on 8% SDS-PAGE gels. Proteins were transferred to a PDVF membrane (Millipore Corp), subjected to Western blotting, 30 blocked for 1 hour with odyssey blocking buffer and incubated with the appropriate antibody 1:500 (vol/vol) in odyssey blocking buffer with 0.1% (vol/vol) Tween 20 (16 hours, 4°C). After washing, membranes were incubated with the appropriate LICOR secondary antibody (1 hour, 4°C).…”