A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase

Hisaaki Kawakatsu, Takao Sakai, Yumiko Takagaki, Yasuhiko Shinoda, Masaki Saito, M. Koji Owada, Junichi Yano

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82 Citations (Scopus)

Abstract

Phosphorylation and dephosphorylation of Tyr-530 in human c-Src (Tyr-527 in avian c-Src) is critical in regulating c-Src kinase activity. So far, it has not been possible to distinguish the active and inactive forms in vivo. We now report a new monoclonal antibody that selectively recognizes the active form of c-Src. This antibody, termed clone 28, recognized a region adjacent to Tyr-530 (Q529YQP532) in the C-terminal regulatory domain of c-Src, and its binding was hindered by phosphorylation of this tyrosine as determined by peptide competition assay. Combined immunoprecipitation/Western blotting revealed that clone 28 reacted with a 60-kDa protein that was precipitated by mAb 327, a well known monoclonal antibody against v-Src and c-Src. Cyanogen bromide cleavage and two-dimensional tryptic maps confirmed that clone 28 was specific for the active form (Tyr-330 not phosphorylated), whereas mAb 327 recognized the inactive form (Tyr-530 phosphorylated) as well as the active form. Clone 28 selectively immunoprecipitated the active form and augmented its kinase activity. Preabsorption experiments revealed that clone 28 could not completely immunoprecipitate the mAb 327 binding 60-kDa protein in either an in vitro or an in vivo phosphorylation system. These observations, taken together, strongly suggest the existence of multiple forms of c-Src as proposed by Cooper and Howell (1993) (Cooper, J. A., and Howell, B. (1993) Cell 73, 1051-1054). Using clone 28, we demonstrated a distinct localization of the active form of c-Src within cultured normal fibroblast cells. In liver tissue sections, we also examined the distribution of the active form in embryonic mice. Megakaryocytes were strongly stained, in contrast to completely negative immunoreactivity in hepatocytes, reticulocytes, and granulocytes. This result provides the first direct evidence that c-Src is highly activated in platelets.

Original languageEnglish
Pages (from-to)5680-5685
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number10
DOIs
Publication statusPublished - 08-03-1996
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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