Identification of PINK1 Substrates Using Conditional PINK1 Knockout Cells and Quantitative Phosphoproteomics

I. Abstract and Si2nificance OLPrODOSd Prolect Mutations in the PARK6 (PINK1) gene cause early-onset recessive Parkinson's disease (PD). PINK! is a mitochondrial serine/threonine kinase, whose deficiency !eads to mitochondria! abnormalities and dopamine neuron and muscle degeneration in flies. PINK1 is believed to phosphory!ate specific proteins (substrates) that are necessary to maintain mitochondria! homeostasis and to protect cells against endogenous and exogenous stress. We hypothesize that abolishing cellular PINK1 kinase activity will result in decreased or absent phosphorylation of PINK1 substrates. To investigate this, we have generated mouse embryonic stem cells with a conditional PINK! gene knockout. The aim of the proposed study is to identify novel PINKJ substrates by comparing the abundance of phosphoproteins in these cells before and after deletion of the PINK1 gene by Cre-mediated recombination using quantitative mass spectrometry. Uncovering new PINK! substrates will increase our understanding of the molecular mechanisms underlying dopamine neuron death and may reveal new targets for PD therapy.