title: Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.
abstract: SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.
authors.affiliation: Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Ritzerfeld
authors.forename: Steffen
authors.initials: S
authors.lastname: Remmele
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Koen
authors.initials: K
authors.lastname: Temmerman
authors.forename: Britta
authors.initials: B
authors.lastname: Brügger
authors.forename: Sabine
authors.initials: S
authors.lastname: Wegehingel
authors.forename: Stella
authors.initials: S
authors.lastname: Tournaviti
authors.forename: Jeroen R P M
authors.initials: JR
authors.lastname: Strating
authors.forename: Felix T
authors.initials: FT
authors.lastname: Wieland
authors.forename: Beate
authors.initials: B
authors.lastname: Neumann
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
authors.forename: Chris
authors.initials: C
authors.lastname: Lawerenz
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hesser
authors.forename: Holger
authors.initials: H
authors.lastname: Erfle
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
authors.forename: Walter
authors.initials: W
authors.lastname: Nickel
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology*; D020756:Coatomer Protein / Q000235:genetics / Q000378:metabolism; D015894:Genome, Human; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006706:Homeostasis; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D050356:Lipid Metabolism; D054878:Lipoylation; D010641:Phenotype; D054730:Protein Interaction Domains and Motifs; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051575:Proto-Oncogene Proteins c-yes / Q000378:metabolism; D034741:RNA, Small Interfering; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D020756:Coatomer Protein; D034741:RNA, Small Interfering; D051575:Proto-Oncogene Proteins c-yes; C489834:YES1 protein, human; D019061:src-Family Kinases; D051571:Protein Kinase C-alpha