Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn

Tyrosine kinases, c-Src and Fyn, in their active form, have their C-termina l tyrosine residue dephosphorylated. In this study, we used clone 28, a mon oclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosin e of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin alpha IIb beta 3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 mu M TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Tr iton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Fi ve minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskele ton, while only about 20% of c-Src was recovered in this fraction. The Trit on-insoluble fraction was further fractionated by RIPA (radioimmunoprecipit ation assay) buffer containing 0.1% SDS. While active c-Src was predominant ly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negati ve c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fra ction. These findings suggest that the moot of activation and redistributio n into the cytoskeleton differs between c-Src and Fyn, and that clone 28 pr ovides a useful tool for investigating the activation and mobilization of S rc family tyrosine kinases. (C) 2000 Elsevier Science B.V. All rights reser ved.