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 IntroductionPlatelet outside-in signaling is an important process in hemostasis and refers to the process of fibrinogen binding to ␣IIb␤3, an integrin only expressed in platelets and megakaryocytes. Upon fibrinogen binding, integrin ␣IIb␤3 undergoes a conformational change that initiates signaling cascades that modulate the actin cytoskeleton leading to cell spreading, filopodia and lamellipodia formation and at later stages, cell retraction. 1 Coordinated spreading and retraction is of pivotal importance in platelets as stable platelet adhesion and thrombus formation depends on it. Engagement of ␣IIb␤3 results in tyrosine phosphorylation of proteins, therefore the role of tyrosine kinases in platelet outside-in signaling has been extensively studied. A hierarchical activation of Src-family kinases (SFK) and Spleen Tyrosine kinase (Syk) has been proposed as a general mechanism for integrin signaling. 2 Of the members of SFK expressed in platelets both Src and Fyn have been reported to interact with ␤3 cytoplasmic tail although at distinct sites and with different functional consequences. Src constitutively associates with the RGT sequence on the ␤3 tail, 3 becomes activated upon fibrinogen binding and its further removed by a calpain-dependent cleavage. 1 Fyn associates with the IHDRK sequence on the ␤3 tail, has a sustained interaction with the integrin through out the platelet activation process, and is postulated as an alternative pathway to support platelet-spreading. 4 Syk, on the other hand, is tyrosine phosphorylated in a SFK-dependent manner. 5,6 The activation of Src kinase and Syk by ␣IIb␤3 results in tyrosine phosphorylation of signaling molecules and adaptor proteins that are implicated in modulation of the actin cytoskeleton. 7,8 Recently, the involvement of ITAM motifs of the Fc␥RIIA receptor has also been described in the human platelet outside-in signaling complex, 9 which suggests the possibility that integrins signal as immunoreceptors. In this context, the activation of Syk by ␣IIb␤3 is mediated by...

The N-terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets

“…Clot retraction was not altered in blood from Syk R41Afl/fl;PF4-Cre mice or in blood treated with a Syk inhibitor ( Figure 5C), consistent with previous reports that Syk is not involved in this response. 18 Whole-cell tyrosine phosphorylation was not altered in Syk R41Afl/fl;PF4-Cre platelets that had been allowed to spread on fibrinogen, whereas treatment with the Syk and Src inhibitors, PRT318 and PP2, partially and completely inhibited phosphorylation, respectively (Figure 5B). The inhibitors had a similar effect on platelet spreading (not shown).…”

“…[13][14][15] The 2 conserved tyrosines of the b3-cytoplasmic tail do not form an ITAM and, upon phosphorylation, mediate clot retraction through recruitment of myosin II, 16,17 a process that is independent of Syk. 18 The b3 C-terminal tail undergoes constitutive association with Src, which lies upstream of Syk. 19 A functional role for Src and Syk in outside-in signaling by aIIbb3 in mice platelets was shown using Src family kinase-deficient and Syk-deficient mice.…”

Inhibition of SRC Corrects GM-CSF Hypersensitivity That Underlies Juvenile Myelomonocytic Leukemia

“…These observations suggest that in addition to gene mutation-induced conformational changes, Src-dependent phosphorylation contributes to Cbl binding to GMRbc. Src has been shown to activate and phosphorylate Cbl on multiple tyrosine residues, including Y700, 731, and 774, which promote several signaling cascades (30). Src also phosphorylates Cbl on Y371 to activate the E3 ligase function of Cbl, which in turn trigger interdependent ubiquitylation and degradation of both proteins (26), a process necessary for terminating downstream signaling postactivation.…”

Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy

“…Platelet receptors and cytoplasmic molecules, such as calpain-1 [20] and talin [21] which are involved in signaling cascades after adhesion and activation have been extensively studied. However, the effect of these cascades on plug morphology have been commonly analyzed through the transmission microscopy alone or combined with fluorescence in 2 dimensions only [11,20,22,23]. The DHM is very convenient to use and gives more information on the effect of the molecules involved in the spreading on the nascent aggregate shape.…”

“…These tests are based on turbidimetric optical detection, multiple electrode aggregometry and flow cytometry [10]. Using such tests, it is impossible to analyze the processes that follow platelets adhesion, namely the spreading and the retraction although they both play a pivotal role in the platelet adhesion and the thrombus formation [11].…”