A New Monoclonal Antibody Which Selectively Recognizes the Active Form of Src Tyrosine Kinase

doi:10.1074/jbc.271.10.5680

A New Monoclonal Antibody Which Selectively Recognizes the Active Form of Src Tyrosine Kinase (*)

From the (1)Molecular Biology Department, Research Laboratories, Nippon Shinyaku Co. Ltd., 601 Kyoto, Japan, the
(2)Division of Hemopoiesis, Institute of Hematology, Jichi Medical School, 329-04 Tochigi, Japan, and the
(3)Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, 607 Kyoto, Japan

^§ To whom correspondence should be addressed. Tel.: 81-75-321-1111 (ext. 8932); Fax: 81-75-313-5001.

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 (QYQP) 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-530 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 fibrobast 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SH

Src homology

mAb

monoclonal antibody

TBS

Tris-buffered saline

PBS

phosphate-buffered saline

RIPA

radioimmunoprecipitation buffer.
↵²T. Sakai and H. Kawakatsu, unpublished observation.
- Received July 18, 1995.