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Genome-wide RNA Interference Study Reveals Gene Products Involved in Intracellular Targeting of Src Kinases
Posted August 01 2011
Collaborative Study of several CellNetwork members published in 'Genome Research'

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 in turn are both necessary and sufficient to mediate specific targeting of Src kinases to the inner leaflet of plasma membranes. Acylation of most Src kinases proceeds in two steps with co-translational N-terminal myristoylation being followed by palmitoylation of cysteine residues, a process that occurs at the cytoplasmic leaflet of Golgi membranes. Intracellular transport of Src kinases from Golgi membranes to the inner leaflet of the plasma membrane depends on microdomains into which Src kinases partition upon palmitoylation. In the current 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 signalling 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. The complete list of validated gene products demonstrated to be involved in Src kinase targeting represents a major resource for future studies unravelling the molecular mechanisms that underlie proper targeting of Src kinases to the inner leaflet of plasma membranes.

 

Genome Res. 2011 Jul 27. [Epub ahead of print]
Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of Src kinases.
Ritzerfeld J, Remmele S, Wang T, Temmerman K, Brügger B, Wegehingel S, Tournaviti S, Strating JR, Wieland FT, Neumann B, Ellenberg J, Lawerenz C, Hesser J, Erfle H, Pepperkok R, Nickel W.