title: Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells.
abstract: Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-l-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein-protein interactions in cells.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Nobumasa
authors.initials: N
authors.lastname: Hino
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oyama
authors.forename: Aya
authors.initials: A
authors.lastname: Sato
authors.forename: Takahito
authors.initials: T
authors.lastname: Mukai
authors.forename: Fumie
authors.initials: F
authors.lastname: Iraha
authors.forename: Akiko
authors.initials: A
authors.lastname: Hayashi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kozuka-Hata
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001389:Azirines / Q000737:chemistry*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D018909:src Homology Domains
chemical_list: C095516:AFDN protein, human; D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D001389:Azirines; D018797:Cell Cycle Proteins; D003432:Cross-Linking Reagents; C485647:GIT1 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D010649:Phenylalanine; D004815:Epidermal Growth Factor; C044966:3-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)alanine; D009218:Myosins; D016547:Kinesins