Ubiquitin phosphorylated at Ser57 hyper-activates parkin

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Parkin, a ubiquitin (Ub) E3 ligase, is essential for mitophagy and protein quality control, with its malfunction linked to Parkinson's disease.
• Parkin activity is known to be stimulated by ubiquitin phosphorylation at Ser65 (pUb^S65), primarily by the kinase PINK1.
• The functional significance of other ubiquitin phosphorylation sites beyond Ser65 remains largely unexplored.

Purpose of the Study:

• To investigate the biochemical function of different ubiquitin phosphorylation sites on parkin activation.
• To quantitatively assess the impact of ubiquitin phosphorylation site and stoichiometry on parkin's catalytic activity.
• To identify novel pathways regulating parkin activity, particularly PINK1-independent mechanisms.

Main Methods:

• Utilized fluorescently labeled and site-specifically phosphorylated ubiquitin substrates.
• Employed fluorescence measurements to quantify parkin activity in response to various ubiquitin phosphorylation states.
• Analyzed the synthesis of polyubiquitin chains and the ubiquitination of the substrate Miro-1.

Main Results:

• Phosphorylation at Ser65 (pUb^S65) stimulated parkin activity 5-fold compared to un-stimulated parkin.
• Phosphorylation at Ser12 (pUb^S12) showed a low but detectable level of parkin activity.
• Phosphorylation at Ser57 (pUb^S57) dramatically hyper-activated parkin, enabling selective synthesis of poly-pUb^S57 chains even with low pUb^S57 stoichiometry.
• Parkin preferentially ubiquitinated Miro-1 with pUb^S65 and even more efficiently with pUb^S57 chains.

Conclusions:

• Ubiquitin phosphorylation at sites other than Ser65, particularly Ser57, plays a significant role in modulating parkin activity.
• pUb^S57 represents the first identified PINK1-independent activator of parkin, revealing a new regulatory pathway.
• These findings expand our understanding of ubiquitin phosphorylation in governing parkin stimulation and its implications for Parkinson's disease.