Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage

Abstract

Upon activation by different transmembrane receptors, the same signaling protein can induce distinct cellular responses. A way to decipher the mechanisms of such pleiotropic signaling activity is to directly manipulate the decision-making activity that supports the selection between distinct cellular responses. We developed an optogenetic probe (optoSRC) to control SRC signaling, an example of a pleiotropic signaling node, and we demonstrated its ability to generate different acto-adhesive structures (lamellipodia or invadosomes) upon distinct spatio-temporal control of SRC kinase activity. The occurrence of each acto-adhesive structure was simply dictated by the dynamics of optoSRC nanoclusters in adhesive sites, which were dependent on the SH3 and Unique domains of the protein. The different decision-making events regulated by optoSRC dynamics induced distinct downstream signaling pathways, which we characterized using time-resolved proteomic and network analyses. Collectively, by manipulating the molecular mobility of SRC kinase activity, these experiments reveal the pleiotropy-encoding mechanism of SRC signaling.

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

“…Actually, Src-ULBR may act as a fine-tuning mechanism, which may be exacerbated upon Src overactivation to promote cancer development. This fine-tuning mechanism is supported by previous works on the Src capacity to induce Xenopus oocytes maturation (10), Src regulation of retinal ganglion cell survival or postmitotic neuron function by Ser75 phosphorylation (31,32) and is consistent with a recent Src optogenetic study (33).…”

Regulatory Roles of the N-Terminal Intrinsically Disordered Region of Modular Src

“…Kerjouan et al [39] focused on the mechanism of pleiotropic signaling activity. To decipher the ability of Src to generate two different actin-associated structures (i.e., lamellipodia and invadosomes at adhesive sites on the plasma membrane), an optogenetic and photoactivable Src probe (optoSrc) based on a cryptochrome 2 (CRY2) optogenetic module was selected to activate and control Src spatiotemporally [39]. OptoSrc lacks an N-myristoyl membrane-anchoring domain and has a non-phosphotyrosine-binding R175L mutation in SH2 and an open conformation-inducing Y530F mutation.…”

“…OptoSrc lacks an N-myristoyl membrane-anchoring domain and has a non-phosphotyrosine-binding R175L mutation in SH2 and an open conformation-inducing Y530F mutation. OptoSrc, which is not accumulated in any cellular compartment in the dark in Madin-Darby Canine Kidney (MDCK) cells, is directed to generate each adhesive structure by controlling the dynamics of optoSrc dimers/oligomers, which is induced by the ability of CRY2 to oligomerize, in adhesive sites formed by photostimulation [39].…”

“…The different decision-making events regulated by optoSrc dynamics induce the activation of various physiological substrates in Src signaling pathways. Relocalization of the optoSrc dimer to adhesive sites is promoted by SH3 to recognize the PPP of other proteins and to control Src kinase activity [39]. Src dimerization/oligomerization may lead to phase separation induced by SH3 domain multimers [56,74], which might favor SH3 cooperativity that stimulates additional interactions in signaling molecules [39].…”

“…Furthermore, SH4-UD in the IDR regulates dimerization/oligomerization of Src proteins [37] and the modulation of membrane anchoring via myristoylated SH4, which interacts with SH3 domains, as revealed by NMR studies on the IDR of Src [38]. Furthermore, optogenetic research and chemical genetic methods combined with comprehensive mutagenesis has revealed novel aspects of Src molecular behavior and activity controlled by the IDR [19,39]. ).…”