title: Synthesis of functional signaling domains by combinatorial polymerization of phosphorylation motifs.
abstract: The adaptor protein Cas contains a core substrate domain with multiple YXXP motifs that are phosphorylated by Src and other tyrosine kinases. Here, we used a synthetic strategy to determine the importance of the arrangement, spacing, and identity of the YXXP motifs. By polymerizing short DNA sequences encoding two phosphorylation motifs, we created a panel of Cas mutants in which the entire substrate domain was replaced by synthetic domains containing random numbers and arrangements of the motifs. Most of these synthetic Cas variants were recognized and phosphorylated by Src in vitro and in intact mammalian cells. The random polymer mutants also restored migration activity to Cas knockout cells; even artificial proteins containing a single motif retained some biological function. Our results suggest that the arrangement of Cas motifs is not critical for signaling. This method could be used to identify the minimal functional units in other signaling proteins.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
authors.forename: Parag
authors.initials: P
authors.lastname: Patwardhan
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Shiba
authors.forename: Chris
authors.initials: C
authors.lastname: Gordon
authors.forename: Barbara P
authors.initials: BP
authors.lastname: Craddock
authors.forename: Minamisawa
authors.initials: M
authors.lastname: Tamiko
authors.forename: W Todd
authors.initials: WT
authors.lastname: Miller
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D050719:Crk-Associated Substrate Protein; D019061:src-Family Kinases