© 2006 The Japanese Biochemical Society.
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Phylogeny of Vertebrate Src Tyrosine Kinases Revealed by the Epitope Region of mAb327
1 Research Center for Environmental Genomics, 2 Biosignal Research Center, and 4 Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501; 3 CREST, Japan Science and Technology Agency, Kawaguchi 332-0012; 5 Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi, Yokohama 230-0045; and 6 Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, Yamashina, Kyoto 607-8412
* To whom correspondence should be addressed. Tel: +81-78-803-5952, Fax: +81-78-80-5951, E-mail: tiwasaki{at}kobe-u.ac.jp
Mass fingerprinting and MS/MS analysis demonstrated that Xyk, a 57-kDa Src family tyrosine kinase that is activated within minutes of Xenopus egg fertilization, comprises a mixture of two Src proteins, Src1 and Src2. However, the Xenopus Src protein, denoted as xSrc, is hardly detectable with mAb327, a universal Src-specific antibody, whose target sequence has not yet been determined. We show that a point amino acid substitution in the Src homology 3 domain of xSrc is critical for improvement of the low efficiency of its recognition by mAb327. Namely, a point-mutated xSrc, in which Arg-121 was replaced by His that is conserved among mAb327-reactive Src in mammals and chicken, showed increased recognition by mAb327. On the other hand, a mutant chicken Src, in which the His-122 residue is replaced by Arg, showed decreased recognition by mAb327. Genomic sequencing analysis also demonstrated that reptile Src proteins are of either the R-type (snake) or H-type (caiman, turtle, and tortoise). These studies revealed, for the first time, a critical amino acid in the Src SH3 domain for mAb327 recognition, and suggest a novel scheme for the molecular evolution of Src, in which the H-type Src(s) are monophyletic and derived from the R-type Src.
Present address: Hokko Research Laboratories, Hokko Chemical Industry Co., Ltd, 2165, Toda, Atsugi, Kanagawa 243-0023.
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