Taules, M. | bioRxiv Science

bioRxiv2024

Lipid-driven SRC self-association modulates its transformation capacity

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 StatementCondensate 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 Srcs transforming capacity. O_LIFull length Src and truncated variants including its myristoylated SH4 domain self-associate on the surface of homogeneous lipid bilayers. C_LIO_LIThe interaction of a lysine cluster in SH4 domain with lipids modulates Src self-association C_LIO_LIAtomic 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 C_LIO_LISrc self-association is confirmed in human cells and the Src transforming function of Src in human cells is modulated by lipid-mediated self-association C_LI

molecular biology↗

bioRxiv2022

Lipid-mediated dimerization of membrane-anchored c-Src is driven by a cluster of lysine residues in the N-terminal SH4 domain.

The membrane-anchored c-Src tyrosine kinase mediates signaling from a wide range of cell surface receptors controlling cell growth, adhesion, and survival. c-Src deregulation is associated with cancer. Dimerization appears to be a novel layer of regulation through a yet unclear mechanism. Binding of c-Src tyrosine kinase to the plasma membrane is mediated by the myristoylated and strongly positively charged N-terminal SH4 domain. Although activation of c-Src is known to require phosphorylation by a second c-Src molecule, electrostatic repulsion between the charged residues was considered to prevent dimerization. Here we show that a cluster of positively charged lysine residues in c-Src SH4 domain not only does not prevent dimerization but, in fact, enhances it through a lipid-mediated process. Dimerization not only depends on the number of positive charges but also on their position and the nature of the charged residues. Replacement of lysine by arginine increases dimerization in vitro and in vivo and, in HEK293T cells, causes a two-fold increase in tyrosine phosphorylation. Lipid mediated protein-protein interactions induced by clusters of basic residues may represent a general mechanism for modulating cell signaling, consistent with the abundance of positively charged residues in the juxta membrane region of many signaling proteins.

biophysics↗