title: 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.
authors.affiliation: Department of Physiology, Temple University, Philadelphia, PA, USA.
authors.forename: Lorena
authors.initials: L
authors.lastname: Buitrago
authors.forename: Wallace Y
authors.initials: WY
authors.lastname: Langdon
authors.forename: Archana
authors.initials: A
authors.lastname: Sanjay
authors.forename: Satya P
authors.initials: SP
authors.lastname: Kunapuli
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000502:physiology*; D003018:Clot Retraction / Q000502:physiology*; D006487:Hemostasis / Q000502:physiology*; D006801:Humans; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D011505:Protein-Tyrosine Kinases; C067419:CBL protein, human; C493849:Cbl protein, mouse