Lipid-driven SRC self-association modulates its transformation capacity

This is the authors' abstract. We don't add key points for this paper.

Src tyrosine kinase is anchored to the plasma membrane, however, the contribution of membrane lipids to its regulation remains elusive. Here we report Src self-association through a lysine cluster in the Src SH4 region mediated by lipids in human cells and in vitro. Mutation of the lysine cluster to arginine modulates Src self-association and its transforming function in human cells. This mechanism may also apply to other membrane-associated signaling proteins that have similar lysine clusters in their unstructured regions. Lipid-anchored micron-sized condensates of full length Src are formed in supported homogeneous lipid bilayers (i.e independently of lipid phase separation). Condensates are also formed by the purified Src N-terminal regulatory element, including the myristoylated SH4 domain, the intrinsically disordered Unique domain and the globular SH3 domain. The isolated SH4 domain forms small protein-lipid clusters, but not micron-size condensates. Our findings reveal lipid-mediated kinase self-association as an additional mechanism for Src regulation. Significance Statement Condensate formation, often associated to intrinsically disordered protein regions is emerging as a widespread regulatory mechanism. Protein phase separation has been linked to the regulation of kinases recruited to condensates formed by other proteins. Here we show i) that the N-terminal disordered region of Src kinase drives lipid-mediated self-association to form condensates on the cell surface, ii) a lysine cluster in the N-terminal SH4 domain is essential for this process, iii) mutation of these lysine residues to arginine increases self-association in vitro and in vivo, and iv) self-association of Src results in enhanced transforming capacity in human cells. Similar lysine clusters are present in juxtamembrane regions suggest a general mechanism for modulating protein-protein interactions by lipids. The article shows the formation of condensates by Src on supported lipid membranes mediated by lipids, dependent of a conserved lysine cluster in its SH4 domain, and modulating Src’s transforming capacity. Full length Src and truncated variants including its myristoylated SH4 domain self-associate on the surface of homogeneous lipid bilayers. The interaction of a lysine cluster in SH4 domain with lipids modulates Src self-association Atomic force microscopy shows the formation of micron-size condensates and smaller clusters on the surface of supported lipid bilayers, depending on the lipid composition or the presence of mutations in the lysine cluster Src self-association is confirmed in human cells and the Src transforming function of Src in human cells is modulated by lipid-mediated self-association