Csk binding to integrin β3 is regulated by tyrosine and threonine phosphorylation of β3

C-terminal Src kinase (Csk) is crucial for the normal function of platelet integrin αIIbβ3 because it inactivates Src kinase. While the molecular mechanism by which cytoplasmic Csk associates with membrane-bound Src is known in resting platelets, it remains unknown in activated platelets. Using surface plasmon resonance, a kinase assay, and microscale thermophoresis, we discovered that Csk binds directly to phospho-Tyr747 on the β3 tail via its SH2 domain and that this binding fully activates Csk. Moreover, we found that phospho-Thr753 on the β3 tail prevents Csk from binding. Collectively, our findings provide new insight into the regulation of Csk function in activated platelets, suggesting that Tyr747-phosphorylated β3 recruits Csk near active Src, whereas phospho-Thr753 on β3 inhibits this interaction.