Abstract
This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phos-tag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.
Keywords: PINK1, Parkin, Ubiquitin, Mitophagy, Parkinson's disease
Background
In cells, PINK1 is stabilized and activated by mitochondrial membrane depolarization and other forms of stress that lead to mitochondrial damage. Activated PINK1 phosphorylates ubiquitin, which acts as the receptor for the cytosolic E3 ubiquitin ligase Parkin on the mitochondrial surface. Phosphorylation of Parkin by PINK1 is required for full activity of Parkin towards mitochondrial substrates. The presence of active pS65-Parkin amplifies in a feed-forward mechanism the amount of pS65-ubiquitin on mitochondria, which acts as the mitophagy tag. Eventually, damaged mitochondria are being recognized by autophagy adapters and will be degraded by the proteasome and by autophagy (mitophagy). This crucial mitochondrial quality control pathway promotes turnover of mitochondria and prevents accumulation of dysfunctional mitochondria that can lead to cellular degeneration. Loss-of-function mutations in either PINK1 or Parkin are associated with early-onset Parkinson’s disease. In cell-free ubiquitination assays, the presence of pS65-Ub activates the E3 ligase activity of Parkin also in the absence of active PINK1. While many groups use their own purified recombinant proteins for in vitro PINK1 kinase assays, this optimized protocol describes the in vitro phosphorylation of ubiquitin and Parkin with commercially available recombinant proteins.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The here described method uses Western blot analysis as read-out. Phosphorylation by PINK1 over time (Figure 1) can be quantified using densitometric analysis of band intensities using open source software (e.g., ImageJ or ImageStudio Lite). Reactions performed with mutant PINK1 D359A serve as a negative control. On phos-tag gels (Figure 1) the ratio of the shifted versus the non-shifted band allows to calculate the ratio of modified to non-modified substrate. Statistical analysis should be performed with the results from at least three independent experiments. Figure 1. Kinase assay with N-terminally tagged monomeric ubiquitin using wild-type (WT) or kinase-dead (KD) MBP-TcPINK1. Phosphorylated ubiquitin was detected on a 14% Tris-glycine phos-tag gel with HRP-coupled streptavidin (upper panel) and on a standard 16% Tris-glycine gel with a phospho-specific ubiquitin antibody (pS65-Ub#2) (lower panel).
Recipes
Acknowledgments
This protocol was adapted from Fiesel et al. (2015) and Fiesel and Springer (2015). FCF is the recipient of a fellowship from the American Parkinson’s Disease Association. WS was partly supported by the NIH/NINDS R01NS085070, the Michael J. Fox Foundation for Parkinson’s Research, the Foundation for Mitochondrial Medicine, the Mayo Clinic Foundation, the Center for Individualized Medicine, the Center for Regenerative Medicine, the Center for Biomedical Discovery, the Marriott Family Foundation, and a Gerstner Family Career Development Award. WS is a member of a Neuroscience-Focused Research Team at Mayo Clinic Florida.
References
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