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.

High-Content siRNA Screen Reveals Global ENaC Regulators and Potential Cystic Fibrosis Therapy Targets

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HIV-1 Nef disrupts membrane-microdomain-associated anterograde transport for plasma membrane delivery of selected Src family kinases

“…Notably, phosphorylation of N‐terminal serines by protein kinase C (PKC) isoforms enhances membrane interactions and biological activity of Lck (Yasuda et al ., ) as well as Nef (Wolf et al ., ). We recently identified PKCalpha as a critical component of PM delivery of the SH4 domains of the Leishmania protein HASPB and the SFK Yes (Ritzerfeld et al ., ), suggesting PKC‐mediated phosphorylation as a general regulatory mechanism of SH4 domain membrane interactions and intracellular transport. Moreover, SH4 domain trafficking appears to be regulated by dynamic phosphorylation/dephosphorylation cycles (Tournaviti et al ., ) and the outcome of this regulation may be SH4 domain specific as a function of, e.g.…”

Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion

“…While attempts to screen Leishmania genetically for effectors in this process have been challenging, a recent human genome-wide RNAi screen for factors involved in SH4-dependent protein targeting may be informative. Using SH4-HASPB–GFP and a mammalian SRC kinase YES-1 reporter (SH4-YES1-mCherry), 13 gene products were identified that may function primarily in HASPB targeting and a further 23 that are required for both HASPB and YES1 targeting ( Ritzerfeld et al ., 2011 ). Those with high scores in both HASPB and YES1 intracellular trafficking included COPB1 (a subunit of the coatomer complex), protein kinase C, the membrane receptors ITGAV (integrin alpha vitronectin) and ITGB1 (integrin beta 1), and PRP8 (pre-mRNA processing factor 8 homologue).…”