title: Reengineering the signaling properties of a Src family kinase.
abstract: Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expressed in mammalian cells, the substrate specificity of the PDZ-kinases was directed to a different group of proteins than wild-type Hck. The PDZ-kinases phosphorylate neuronal nitric oxide synthase (nNOS), a known binding partner of the syntrophin PDZ domain. We also introduced a PDZ ligand at the C-terminus of the adaptor protein Cas. PDZ-Hck kinases phosphorylate the engineered Cas protein in Cas(-/-) cells and restore the migration defect of these cells. A PDZ-kinase was also functional in rewiring MAPK signaling via an engineered ErbB2 construct containing a PDZ ligand sequence. Several of the PDZ-kinases show autoregulatory properties similar to natural SFKs. Thus, the PDZ-ligand interaction is able to functionally replace the normal SH2-pY527 interaction that regulates SFKs. Our data highlight the modularity and evolvability of signaling proteins.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Stony Brook University, Stony Brook, New York 11794-8661, USA.
authors.forename: Shalini S
authors.initials: SS
authors.lastname: Yadav
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Yeh
authors.forename: Barbara P
authors.initials: BP
authors.lastname: Craddock
authors.forename: Wendell A
authors.initials: WA
authors.lastname: Lim
authors.forename: W Todd
authors.initials: WT
authors.lastname: Miller
mesh_terms: D000818:Animals; D019556:COS Cells; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000235:genetics; D002522:Chlorocebus aethiops; D050719:Crk-Associated Substrate Protein / Q000235:genetics / Q000378:metabolism; D049029:Dystrophin-Associated Proteins / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D054731:PDZ Domains / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D051572:Proto-Oncogene Proteins c-hck / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics
chemical_list: D050719:Crk-Associated Substrate Protein; D049029:Dystrophin-Associated Proteins; D052248:Nitric Oxide Synthase Type I; D051572:Proto-Oncogene Proteins c-hck; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C091824:syntrophin; C496321:Nos1 protein, mouse