title: Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms.
abstract: The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases' homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.
authors.affiliation: Department of Pharmacology.
authors.forename: Pei-Hsuan
authors.initials: PH
authors.lastname: Chu
authors.affiliation: Department of Pharmacology.
authors.forename: Denis
authors.initials: D
authors.lastname: Tsygankov
authors.affiliation: Department of Biomedical Engineering.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Berginski
authors.affiliation: Department of Pharmacology, Department of Biochemistry and Biophysics, and.
authors.forename: Onur
authors.initials: O
authors.lastname: Dagliyan
authors.affiliation: Department of Biomedical Engineering.
authors.forename: Shawn M
authors.identifier: http://orcid.org/0000-0002-8251-4552
authors.initials: SM
authors.lastname: Gomez
authors.affiliation: Department of Pharmacology.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Elston
authors.affiliation: Department of Pharmacology, khahn@med.unc.edu karginov@uic.edu.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Karginov
authors.affiliation: Department of Pharmacology, Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 khahn@med.unc.edu karginov@uic.edu.
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D055592:Biophysical Phenomena; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D015202:Protein Engineering; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D022061:Tacrolimus Binding Protein 1A
keywords: image analysis; live cell imaging; motion classification; protein engineering; rapamycin