Conformational dynamics regulate SHANK3 actin and Rap1 binding

Abstract

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small GTPases. SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins, and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its N-terminal SPN domain. Here, we demonstrate that SHANK3 interacts directly with actin using its SPN domain. Actin binding can be inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3 and loss of SHANK3-actin binding augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for filopodia formation in cancer cells, dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

The Shank/ProSAP N-terminal (SPN) domain of Shank3 regulates targeting to postsynaptic sites and postsynaptic signalling

“…8A, B). We noted that the pattern observed here for the ΔSPN Shank3 protein is very similar to the distribution observed with another Shank3 variant, namely N52R, an artificial mutant that was shown to induce opening of the SPN-Ank tandem (24). Results for expression of this mutant are included here for comparison (Fig.…”

“…Results for expression of this mutant are included here for comparison (Fig. 8B), again showing Shank3 clusters adjacent to the dendritic shaft and some clusters on thin, long protrusions, as observed before (24). On the other hand, we expressed a Q106P mutant construct.…”

“…The αCaMKII protein must be in its non-phosphorylated, inactive form, and the SPN-Ank tandem must be in a closed conformation for binding to occur (22,23). On the other hand, F-actin binds to the SPN domain when the SPN-Ank tandem is in an open conformation (24). It is currently not clear whether some kind of regulated opening of the closed SPN-Ank conformation occurs in vivo.…”

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders

“…The change of Asn52 to Arg in the SPN domain has been designed based on the 3D-structure, with the intention to weaken the affinity of the SPN to the Ank domain. Indeed we have shown before that it truly opens up the SPN-Ank interaction (30). Upon coexpression of WT and mutant forms of the complete N-terminus with the GFP-tagged SPN domain, only the N52R positive control induced a significant increase in binding of the SPN domain to the N-terminus (Fig.…”

Unravelling cell migration: defining movement from the cell surface

“…We still lack detailed knowledge of how the spatiotemporal signaling variations influence actin cortex reorganization at the molecular level and the mechanical changes happening at the cellular scale [ 188 ]. More research about the actin/adhesion crosstalk is also required [ 359 ]. Buracco and colleagues [ 178 ] argued that we still lack a coherent picture of the global mechanisms that induce a heterogeneous actin distribution within cells.…”