title: Comparative analysis of human SRC-family kinase substrate specificity in vitro.
abstract: Src family kinases (SFKs) are the earliest known family of tyrosine kinases and are widely thought to play essential roles in cellular signal transduction. Although numerous functional analyses have been performed, no study has analyzed the specificity of all SFKs on an equal platform. To gain a better understanding of SFK phosphorylation, we designed a high-throughput in vitro kinase assay on the subproteome scale using surface plasmon resonance. We reacted each of the 11 human SFKs with 519 substrate proteins, and significant phosphorylation was detected in 33.6% (1921) of the total 5709 kinase-substrate combinations. A large number of novel phosphorylations were included among them. Many substrates were shown to be phosphorylated by multiple SFKs, which might reflect functional complementarity of SFKs. Clustering analysis of phosphorylation results grouped substrates into 10 categories, while the similarity of SFK catalytic specificity exhibited no significant correlation with that of amino acid sequences. In silico predictions of SRC-specific phosphorylation sites were not consistent with experimental results, implying some unknown SRC recognition modes. In an attempt to find biologically meaningful novel substrates, phosphorylation data were integrated with annotation data. The extensive in vitro data obtained in this study would provide valuable clues for further understanding SFK-mediated signal transduction.
authors.affiliation: Japan Biological Informatics Consortium, Aomi, Koto-ku, Tokyo 135-8073, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Kawamura
authors.forename: Aya
authors.initials: A
authors.lastname: Miura
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Mori
authors.forename: Ai
authors.initials: A
authors.lastname: Wakamatsu
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Yamamoto
authors.forename: Takao
authors.initials: T
authors.lastname: Isogai
authors.forename: Masaki
authors.initials: M
authors.lastname: Matsumoto
authors.forename: Keiichi I
authors.initials: KI
authors.lastname: Nakayama
authors.forename: Tohru
authors.initials: T
authors.lastname: Natsume
authors.forename: Nobuo
authors.initials: N
authors.lastname: Nomura
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D016000:Cluster Analysis; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D019061:src-Family Kinases