TBK1 (TANK-binding kinase 1), a member of the IKK (IκB kinase) kinase family, is a crucial regulator of various cellular processes, including autophagy, immunity, and inflammation.

Autophagy is a constitutive cellular mechanism which involves the degradation and recycling of cellular components, such as organelles and proteins. Autophagy is upregulated in response to diverse stimuli like nutrient deprivation, oxidative stress, and infection. TBK1 emerges as a critical player in autophagy regulation by interacting with key autophagy-related proteins, including OPTN (optineurin).

Our study aims to investigate the downstream targets of TBK1 in the context of metabolic stress and mitophagy. Mitophagy, the selective autophagy of mitochondria, is induced here by subjecting MCF7 cells to glucose starvation, achieved via shifting the cells to galactose-containing media without any glucose in it. Galactose shifting elevates oxidative phosphorylation due to its less efficient ATP generation compared to glucose glycolysis, resulting in increased mitochondrial damage and subsequent mitophagy upregulation. We have observed a notable shift towards a more respiratory state and significant phosphorylation changes in mitochondrial proteins. However, linking these changes to mitophagy remains challenging. To further assess the impact of oxidative conditions, instead of shifting, we are adapting the cells to galactose media. The more oxidative conditions potentially lead to increased mitochondrial turnover, aiding in the detection of mitophagy-related proteins and TBK1-relevant phosphorylation changes.

To overcome the challenges posed by the galactose shifting experiments, we are exploiting mitophagy-inducing pharmaceutical agents such as Antimycin A, Oligomycin A, and CCCP with the addition of a TBK1 inhibitor to investigate the mitophagy-relevant TBK1 phosphoproteome.

So far, the TBK1 phosphoproteome remains unexplored. We anticipate that our investigation will unveil novel downstream targets of TBK1, shedding light on its role in autophagy regulation.