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Background: PINK1 (PTEN-induced putative kinase 1) gene is the causal gene for recessive familial type 6 of
Parkinson’s disease (PARK6), which is an early-onset autosomal recessive inherited neurodegenerative disease. PINK1
has been reported to exert both autophosphorylation and phosphorylation activity, affecting cell damage under
stress and other physiological responses. However, there has been no report on the identification of PINK1
autophosphorylation sites and their physiological functions.
Methods: (1) We adopted mass spectrometry assay to identify the autophosphorylation site of PINK1, and
autoradiography assay was further conducted to confirm this result. (2) Kinase activity assay was used to compare
the kinase activity of both Ser465 mutant PINK1 and disease-causing mutant PINK1. (3) We use Pulse-chase analysis
to measure whether Ser465 may affect PINK1 degradation. (4) Immunocytochemistry staining was used to study the
PINK1 subcellular localization and Parkin transition in subcellular level.
Result: In our study, we identified the 465th serine residue (Ser465) as one of the autophosphorylation sites in
PINK1 protein. The inactivation of Ser465 can decrease the kinase activity of PINK1. Either dissipated or excessive
Ser465 site phosphorylation of PINK1 can slow down its degradation. PINK1 autophosphorylation contributes to the
transit of Parkin to mitochondria, and has no effect on its subcellular localization. PARK6 causal mutations, T313 M
and R492X, display the same characteristics as Ser465A mutation PINK1 protein, such as decreasing PINK1 kinase
activity and affecting its interaction with Parkin.
Conclusion: Ser465 was identified as one of the autophosphorylation sites of PINK1, which affected PINK1 kinase
activity. In addition, Ser465 is involved in the degradation of PINK1 and the transit of Parkin to mitochondria.
T313 M and R492X, two novel PARK6 mutations on Thr313 and Arg492, were similar to Ser465 mutation, including
decreasing PINK1 phosphorylation activity and Parkin subcellular localization.