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

It was then interesting to test whether cell stress also leads to changes in the intracellular distribution of TBK1. Treatment with arsenite or heat shock resulted in a partial recruitment of TBK1 to SINT-speckles (Figure 7C) , suggesting that this previously identified interaction (Ryzhakov and Randow, 2007) also occurs in MLOs. To investigate a possible contribution of SINTBAD for the activation of TBK1, SINTBAD and AZI2 double-deficient U2OS cells were treated for various periods with arsenite and TBK1 activation was assessed with a phospho-specific antibody by immunoblotting. Knockout of SINTBAD alone had no effect (data not shown), whereas cells lacking SINTBAD and AZI2 ( Figure S2 ) showed reduced TBK1 phosphorylation ( Figure 7D ). To investigate the contribution of SINTBAD and AZI2 for ULK1-induced TBK1 phosphorylation by an independent experimental approach, cells were transfected to express ULK1 together with SINTBAD and/or AZI2. Immunoblotting revealed that ULK1-triggered TBK1 phosphorylation was further enhanced by SINTBAD and AZI2 ( Figure 7E ), corroborating the finding that both adaptor proteins contribute to control of the TBK1 activation threshold. (B) 293T cells were transfected to express hemagglutinin (HA)-SINTBAD along with GFP-AMBRA1 or GFP-PTPN23. One day later, immunoprecipitation (IP) was performed using GFP-Trap beads. IP and input samples were further analyzed by western blot using appropriate antibodies as indicated.