The membrane targeting and spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct RhoB/RhoD endosome requirements.
Src activation is a tightly regulated process which requires RhoB endosome-associated actin assembly and transit to the cell periphery. We show here that although two other ubiquitous Src family kinases (SFKs) Yes and Fyn also require intact actin filaments for peripheral membrane targeting, they display distinct spatial activation and endosomal requirements. Unlike Src, both Yes and Fyn are constitutively membrane-localized to some extent, and Fyn is present in RhoD-positive endosomes whereas Yes does not visibly colocalize with either of the endosomal markers RhoB or RhoD. By modulating amino acid acceptor sites for palmitoylation in Src, Yes and Fyn, we show that Src S3C/S6C, which is palmitoylated (unlike wild-type Src) behaves in a manner more similar to Fyn, by predominantly colocalizing with RhoD endosomes, and the targeting of both Fyn and Src S3C/S6C is inhibited by siRNA-mediated knockdown of RhoD. Moreover, Fyn C3S/C6S, which is no longer palmitoylated, behaves much more like Src by colocalizing with RhoB endosomes and by requiring RhoB for activation and membrane translocation. These data imply that distinct modes of spatial activation and membrane delivery, at least partly under the control of specific acylation attachment sequences and endosome sub-type requirements, define distinct properties of the three ubiquitously expressed SFKs.
Sandilands
Emma
E
The Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK.
Brunton
Valerie G
VG
Frame
Margaret C
MC
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005786:Gene Expression Regulation; D016391:Genes, src; D051379:Mice; D019308:Palmitic Acid / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D051575:Proto-Oncogene Proteins c-yes / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000302:isolation & purification / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000302:isolation & purification / Q000378:metabolism*
D000199:Actins; D019308:Palmitic Acid; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D016392:Proto-Oncogene Proteins pp60(c-src); C491334:Rhod protein, mouse; D020741:rho GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein