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-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin alphaIIbbeta3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 microM 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 Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (radioimmunoprecipitation assay) buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative 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 fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.
Wu
Y
Y
Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Tamaho, Nakakoma, Yamanashi, Japan.
Ozaki
Y
Y
Inoue
K
K
Satoh
K
K
Ohmori
T
T
Yatomi
Y
Y
Owadab
K
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002458:Cell Fractionation; D006801:Humans; D008969:Molecular Sequence Data; D017830:Octoxynol; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015531:Radioimmunoprecipitation Assay; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; C072032:thrombin receptor peptide (42-55); D014443:Tyrosine; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src)