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The SH2 interactome: Development and utility of a phospho‐tyrosine‐specific yeast two‐hybrid system to identify and analyse signalling pathways

We have developed a phosphotyrosine‐specific yeast two‐hybrid system and used it to identify molecules that interact with phosphorylated substrates of Src family kinases (SFK). Using this method has resulted in the identification of new SFK regulatory mechanisms and novel pathways that emanate from SFKs such as the family member Lyn. We show that the Lyn substrate Csk binding protein (Cbp) binds Lyn via a high affinity pY‐SH2 interaction. Lyn phosphorylates Cbp after activation on multiple sites, including a site that recruit Lyn's negative site Y508 phosphorylating kinase, Csk/Ctk, allowing down regulation of Lyn's kinase activity. We also identified the SH2 domain of suppressor of cytokine signaling‐1 (SOCS1) as binding specifically to phosphorylated Y314 of Cbp. We show SOCS1, which is transcriptionally induced by Lyn kinase activity, associates with Cbp to allow the ubiquitination and subsequent proteasomal degradation of Lyn. Further, other pY‐motifs of Cbp interact with SH2 containing mediators of the Akt/PKB pathway. Our system has also identified novel oncogenic substrates of Lyn (Lyn associated cytoskeletal modulator, LACM) that link into the Vav and Nck pathways leading to major changes in cell shape. This new method is amenable to a high throughput screening strategy to identify pathways intersected by all the 120 SH2 domains present in the mammalian genome.