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

Membraneless organelles (MLOs) are liquid-like subcellular compartments providing spatiotemporal control to biological processes. This study reveals that cellular stress leads to the incorporation of the adaptor protein SINTBAD (TBKBP1) into membraneless, cytosolic speckles. Determination of the interactome identified >100 proteins forming constitutive and stress-inducible members of an MLO that we termed SINT-speckles. SINT-speckles partially colocalize with activated TBK1, and deletion of SINTBAD and the SINT-speckle component AZI2 leads to impaired TBK1 phosphorylation. Dynamic formation of SINT-speckles is positively controlled by the acetyltransferase KAT2A (GCN5) and antagonized by heat shock protein-mediated chaperone activity. SINT-speckle formation is also inhibited by the autophagy-initiating kinases ULK1/2, and knockdown of these kinases prevented focal TBK1 phosphorylation in a pathway-specific manner. The phlebovirus-encoded non-structural protein S enhances ULK1-mediated TBK1 phosphorylation and shows a stress-induced translocation to SINT-speckles, raising the possibility that viruses can also target this signaling hub to manipulate host cell functions.