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Abstract:
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. Despite extensive testing of multiple GEF
domain fragments, we did not detect any nucleotide exchange activity in
vitro against 9 representative small GTPases. Bioinformatic analyses
show that obscurin differs from other Trio-subfamily GEFs in several
important aspects. While further research is necessary to evaluate
obscurin GEF activity in vivo, our results indicate that obscurin has
atypical GEF domains that, if catalytically active at all, are subject
to complex regulation.
