ULK1/2 Restricts the Formation of Inducible SINT-Speckles, Membraneless Organelles Controlling the Threshold of TBK1 Activation

Saul, Vera Vivian; Seibert, Markus; Krüger, Marcus; Jeratsch, Sylvia; Kracht, Michael; Schmitz, Michael Lienhard

SINTBAD together with AZI2 controls the threshold of TBK1 phosphorylation, as revealed by loss-of-function and gain-of-function experiments. Active and phosphorylated TBK1 in arsenite-treated cells was largely occurring in the cytosol and showed considerable colocalization with SINT-speckles. In contrast, heat shock-induced TBK1 phosphorylation was mainly nuclear and showed only a partial overlap with SINT-speckles. These differential intracellular localizations together with their distinct dependency on upstream UKL1/2 signals suggest that several pathways lead to TBK1 phosphorylation. Thus it is conceivable that SINT-speckles serve as sites of TBK1 phosphorylation in a stimulus-and context-specific manner. The occurrence of phosphorylated TBK1 in SINT-speckles and also outside from these MLOs can be explained by the fact that only a fraction of TBK1 is found in SINT-speckles at a given time point. A further possible explanation is derived from the mechanism of TBK1 activation, where the initial activation of the kinase leads to rapid interdimer trans-autophosphorylation of its activation loop (Ma et al., 2012) . This implies that after primary activation of the kinase (probably facilitated by the high local protein density in SINTspeckles) active TBK1 can rapidly spread to create high local concentrations at substrate sides. This local enrichment of phosphorylated TBK1 is frequently seen by immunofluorescence and can occur in diverse subcellular localizations (Moharir et al., 2018; Pourcelot et al., 2016) . The localization of TBK1 is also controlled by differential interaction with adaptor proteins including SINTBAD, TANK, and AZI2, which compete for binding to a C-terminal interaction domain in TBK1 (Goncalves et al., 2011) . Contrary to the initial assumption that the TBK1 adaptor proteins control the antiviral function of the kinase, recent publications have shown their dispensability for IRF3 activation (Fang et al., 2017) .