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 have conducted an unbiased RNAi screening approach analyzing the entire human genome for factors involved in proper targeting of SH4 proteins. We have established an experimental model system based on two different SH4 domains that are fused to GFP and mCherry, respectively, and are expressed simultaneously in HeLa cells in a doxycycline-dependent manner. Under control conditions both of these SH4 fusion proteins primarily localize to plasma membranes with only a minor fraction being detected intracellularly. For both primary screening and validation experiments, cells were grown on siRNA arrays. A software tool was developed to detect and quantify perinuclear accumulation of one or both SH4 reporter molecules. Hits identified during primary screening were validated using independent siRNAs.
