Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro.

Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798.