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

As expression of ULK1 can trigger TBK1 phosphorylation , we tested the effect of ULK1 expression on the localization of phosphorylated TBK1. ULK1-expressing cells lacked any areas with focal TBK1 phosphorylation, as the activated kinase was found in the cytosol ( Figure 7A ), revealing that ULK1 can control the distribution of phosphorylated TBK1. To test whether loss of ULK1 and ULK2 affects TBK1 phosphorylation in response to thermal stress, the expression of both kinases was downregulated with specific siRNAs ( Figure S11 ). Immunofluorescence analysis of control cells revealed the heat shock-activated TBK1 in focal structures mainly in the nucleus with a partial overlap with cytosolic SINT-speckles. Downregulation of ULK1/2 largely inhibited the heat shock-induced phosphorylation of TBK1 ( Figure 7B ), suggesting an important contribution of these kinases for this activation pathway. In contrast, arsenite-induced TBK1 phosphorylation was not changed by ULK1/2 knockdown and occurred to a significant part in SINTspeckles ( Figure 7B) , showing that the ULK kinases control phosphorylation of TBK1 in a pathway-specific manner.