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

an independent experimental approach we performed coimmunoprecipitation experiments. These experiments confirmed the results of the mass spectrometry analysis and are exemplified for the interaction of SINTBAD and the tyrosine phosphatase PTPN23 or the autophagy regulator AMBRA1 ( Figure 5B ). Also, binding of ABIN2 (A20-binding inhibitor of nuclear factor-kB activation 2, also referred to as TNIP2) to SINTBAD was confirmed, and mapping experiments revealed the importance of the N-terminal region of SINTBAD for this interaction ( Figure 5C ). Interestingly, expression of SINTBAD and all of its mutants led to an increase in ABIN2 protein levels by an unknown mechanism. The other tested interactions are listed in Table S1 . The identified interacting proteins are known to serve various biological functions, as displayed in Figure 5D . While the biggest group is represented by enzymes and regulators of metabolic processes, two very prominent groups represent proteins with relevance in membrane trafficking or autophagy and also mitosis and cytoskeleton dynamics. The two smallest groups are represented by regulators of innate immunity and proteins mediating transcription, probably assisting in expression of inflammatory gene expression. To test whether mutual interactions have already been described for some of the SINTBAD-interacting proteins, we analyzed the interactors in the STRING database. This analysis revealed the existence of known protein interaction networks between the 150 proteins, as visualized in Figure 5E . We then compared the intracellular localization of SINTBAD with that of some of its interacting proteins. These experiments revealed that some of the proteins such as PTPN23 exactly mirrored the behavior of SINTBAD, as shown in Figure 5F . PTPN23 localizes in the cytosol of control cells and forms speckles after induction of cell stress. Costaining with SINTBAD revealed a complete colocalization in speckles after treatment with heat shock ( Figure 5F ) or arsenite ( Figure S8B ), confirming PTPN23 as a bona fide constituent of speckles. According to the ability of SINTBAD to bind and colocalize with further proteins we term these speckles inducible SINT-speckles.