Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation

Waddell, A.; Ojha, H.; Agarwal, S.; Clarke, C.; Terriente-Felix, A.; Zhou, H.; Kakade, P.; Knebel, A.; Shaw, A.; Gourlay, R.; Varghese, J.; Soares, R.; Toth, R.; Macartney, T.; Eyers, P.; Morrice, N.; Bayliss, R.; Whitworth, A. J.; Eyers, C.; Muqit, M.

doi:10.1101/2023.03.31.534916

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bioRxiv · 10.1101/2023.03.31.534916

Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation

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Abstract

Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinsons disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1.

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Waddell, A., Ojha, H., Agarwal, S., Clarke, C., Terriente-Felix, A., Zhou, H., Kakade, P., Knebel, A., Shaw, A., Gourlay, R., Varghese, J., Soares, R., Toth, R., Macartney, T., Eyers, P., Morrice, N., Bayliss, R., Whitworth, A. J., Eyers, C., Muqit, M.. 2023-04-01. Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation. https://doi.org/10.1101/2023.03.31.534916

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