We found that artificial activation and ligand occupancy of the integrin is enough to dissociate JAM-A from the integrin (Figure 1I). Open in a separate window Figure 1 JAM-A interacts with the integrin IIb3. integrin-associated c-Src in an inactive state by phosphorylating Y529 in its regulatory domain. Absence of JAM-A results in impaired c-SrcY529 phosphorylation and augmentation of outside-in signaling-dependent c-Src activation. Our results strongly suggest that tyrosine-phosphorylated JAM-A is a Csk-binding protein and functions as an endogenous inhibitor of integrin signaling. JAM-A recruits Csk to the integrin-cCSrc complex, where Csk negatively regulates c-Src activation, thereby suppressing the initiation of outside-in signaling. Upon agonist stimulation, JAM-A is dephosphorylated on the tyrosine, allowing the dissociation of Csk from the integrin complex, and thus facilitating outside-in signaling. Introduction On circulating platelets, integrin IIb3 is in a resting conformation incapable of binding its ligand fibrinogen (Fg). During platelet activation by physiological agonists, SKF 86002 Dihydrochloride the integrin is rapidly converted to a high-affinity state through a chain of signaling events termed inside-out signaling. Engagement of activated integrin by Fg transmits signals within the platelets through a cascade termed as outside-in signaling that results in platelet adhesion, spreading, and clot retraction.1 Integrin activation and signaling is important for the process of hemostasis and thrombosis.2,3 Unwanted activation and Rabbit Polyclonal to Collagen V alpha2 signaling can result in life-threatening complications. An elaborate mechanism of regulation of agonist-dependent integrin affinity modulation avoids uninstigated activation of integrin. Likewise, signaling through the integrin (outside-in signaling) is also tightly regulated by both positive and negative regulators. Although a significant amount of SKF 86002 Dihydrochloride information is available on the positive regulators, very little is known about the negative regulators.4 The mechanism of integrin outside-in signaling in platelets is just beginning to be understood. It has been shown that c-Src, a member of the SKF 86002 Dihydrochloride Src family kinases (SFKs), plays a significant role in integrin outside-in signaling.3,5-7 Under resting conditions, SFKs and c-Src are maintained in an inactive state through two intramolecular interactions, including the binding of the Src homology 3 (SH3) domain to a polyproline type II helix,8-10 and the subsequent binding through its Src homology 2 (SH2) domain to a phosphotyrosine residue (Y529) in the C-terminal regulatory domain.11 Maintaining SFKs in an inactive state requires phosphorylation of the C-terminal inhibitory Y529 residue in the SFK by cytoplasmic kinases Csk (C-terminal Src kinase) or Csk homologous kinase.12 It has been shown that Csk suppression of c-Src involves the movement of Csk to sites of c-Src activity.13 Csk also has a SH2 domain through which it binds to a phosphotyrosine residue in a Csk-binding protein.14 Previous studies have shown that the adapter protein, Paxillin, can participate in feedback inhibition of nearby SFKs by recruiting Csk.15,16 It has also been shown that Hic-5, a member of the Paxillin family, is expressed in human platelets and is tyrosine phosphorylated upon platelet activation. Csk is shown to bind phosphorylated Hic-5 via its SH2 domain.16 In resting platelets, c-Src is constitutively associated with the integrins,17 and is kept in an inactive state by phosphorylation at c-SrcY529 residue by Csk.7 However, Hic-5 does not associate with the integrin or regulate c-Src activation. What recruits Csk to the integrin-cCSrc complex in inactive platelets is currently unknown. Cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily have been indicated to exert a negative effect on platelet activation.4 Junctional adhesion molecule A (JAM-A) is a member of the cortical thymocyte marker of the (CTX) family SKF 86002 Dihydrochloride of CAMs.18 JAM-A was originally identified as a platelet receptor for a monoclonal antibody, mAb-F11.19 It has been shown that JAM-A is rapidly phosphorylated during platelet activation by physiological agonists in a protein kinase C-dependent manner.19,20 We recently showed that the genetic ablation of murine resulted in a shortened tail bleeding time.21 Furthermore, we showed that the loss of Jam-A resulted in a prothrombotic phenotype as observed by a variety of in vivo thrombosis.