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

Abstract

c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-Cbl(YF/YF) knock-in mice. c-Cbl KO and c-Cbl(YF/YF) platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl(YF/YF) platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.

Figure 1

c-Cbl activation downstream of αIIbβ3. Human platelets (3 × 10⁸ platelets/mL) were plated over fibrinogen (100 μg/mL) coated plates for the indicated time points. Reaction was stopped with cold lysis buffer. Lysates were examined for c-Cbl phosphorylation using phosphospecific antibodies for residues Y700, Y731 and Y774 as indicated. Equal protein loading was detected with anti–c-Cbl antibody. Cells were also treated with convulxin (Cvx) to ascertain tyrosine phosphorylation of Cbl (positive control). All blots are representative of 3 experiments.

Figure 2

Role of SFKs over c-Cbl phosphorylation. (A) To determine the role of SFKs over c-Cbl phosphorylation downstream of αIIbβ3, platelets were incubated for 10 minutes with 10μM of Pan SFKs inhibitor PP2 or its negative control PP3 and then plated over fibrinogen coated plates for the indicated time points. Lysates were analyzed as described on legend from Figure 1. (B) Densitometry analysis of 5 experiments. Bars represent mean ± SEM of the ratio of phosphorylated protein over total protein. Student t test performed (P < .05, no treatment vs PP2).

Figure 3

Effect of Syk inhibition over c-Cbl phosphorylation downstream of αIIbβ3. (A) To determine the role of Syk mediated phosphorylation downstream of αIIbβ3 platelets were incubated for 10 minutes with 2μM of Syk inhibitor OXSI-2 and then plated over fibrinogen coated plates for the indicated time points. Lysates were analyzed as on legend from Figure 1. (B) SFKs p-Y416 was also analyzed to confirm its intact activity under the same experimental conditions. (C) Densitometry analysis of 5 experiments. Bars represent mean ± SEM of the ratio of phosphorylated protein over total protein. Student t test performed (P < .05, no treatment vs OXSI-2 for residues Y700 and Y774; ns: non-significant for residue Y731).

Figure 4

Functional effect of c-Cbl tyrosine phosphorylation on outside-in signaling-mediated events in human platelets. (A) Human platelets (1 × 10⁶ platelets/mL) were treated with PP2 (10μM) or OXSI-2 (2μM) for 10 minutes before be plated on fibrinogen-coated cover slips for the indicated time point. After 3 times washing with PBS, adherent cells were fixed with 3.7% paraformaldehyde and analyzed with DIC imaging. (B) Clot retraction analysis of human platelets treated with PP2 and OXSI-2.

Figure 5

Role of c-Cbl Y731 on platelet-spreading. (A) Platelets from WT, c-Cbl KO, or c-Cbl^YF/YF were treated with apyrase (0.2 unit/mL) and indomethacin (10μM) and allowed to spread over a fibrinogen-coated cover slip for the indicated time point. After fixation with 3.7% paraformaldehyde, platelets were permeabilized with 0.1% Triton X-100 and stained with Rhodamin-palloidin (red). Platelet morphology was analyzed using confocal microscopy. (B) Surface area quantitation of at least 20 platelets. Graph represents mean ± SEM of platelet area. Student t test performed (P < .05, WT vs c-Cbl KO and WT vs c-Cbl^YF/YF). Representative of 3 different experiments.

Figure 6

CD41 expression, integrin αIIbβ3 activation and α granule secretion on c-Cbl KO and c-Cbl ^YF/YF platelets. (A) Washed platelets from WT, c-Cbl KO and c-Cbl^YF/YF mice were analyzed for αIIb expression with CD-41–FITC antibody. (B) WT, c-Cbl KO and c-Cbl^YF/YF were stimulated with the indicated dose of PAR 4 agonist AYPYKF for 15 minutes in the presence of FITC-labeled anti–P-selectin (CD62) to test α-granule secretion or with PE-labeled JON/A antibody to evaluate integrin αIIbβ3 activation (C). Graphs represent mean ± SEM of % positive cells of 3 different experiments.

Figure 7

Physiologic role of c-Cbl Y731 in platelet outside-in signaling. (A) Murine platelets were allowed to adhere to a fibrinogen-coated cover slip. After 3 times washing with PBS, adherent cells were fixed with 3.7% paraformaldehyde and analyzed with DIC imaging. (B) Clot retraction analysis of platelets from c-Cbl KO and c-Cbl^YF/YF animals. (C) Model depicting the signaling events implicated in c-Cbl phosphorylation downstream of αIIbβ3: Fibrinogen binding to αIIbβ3 cause SFKs activation. The active kinases will phosphorylate c-Cbl on Y731 and activate Syk, which in turn, will phosphorylate c-Cbl Y700 and Y774. The phosphorylated tyrosines 700 and 774 will recruit SH2-domain containing proteins that play a role in platelet-spreading, while phosphorylated Y731 recruit molecules important for both platelet-spreading and retraction.