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.