P.Lock and C.L.Abram contributed equally to this workWe describe a method for identifying tyrosine kinase substrates using anti-phosphotyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries. Several potential Src substrates were identified including Fish, which has five SH3 domains and a recently discovered phox homology (PX) domain. Fish is tyrosine-phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells following treatment with several growth factors. Treatment of cells with cytochalasin D also resulted in rapid tyrosine phosphorylation of Fish, concomitant with activation of Src. These data suggest that Fish is involved in signalling by tyrosine kinases, and imply a specialized role in the actin cytoskeleton.

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Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1 ∆ or chs3 ∆ or the vps23 ∆ chs3 ∆ double knockout strain were unable to rescue the yeast cells as compared to vps23 ∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.