Protein kinase A-induced phosphorylation at the Thr154 affects stability of DJ-1

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P><B>Introduction</B></P> <P>Most cases of Parkinson's disease (PD) are sporadic, but genetic variations have been discovered in PD patients. PARK7/DJ-1 is a known cause of early-onset autosomal-recessive PD and is implicated in neuroprotection against oxidative stress. Although several post-translational modifications of DJ-1 have been proposed, phospho-modification of DJ-1 and its functional consequences have been less studied.</P> <P><B>Methods</B></P> <P>Putative phosphorylation sites of DJ-1 were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS analysis). Subsequently, phosphorylation site of DJ-1 was confirmed by <I>in vitro</I> kinase assay and cell-based pull-down assay. Impaired dimer formation of phospho-null mutant was measured using DSS crosslinking assay and immunoprecipitation assay. To evaluate physiological consequences of this event, protein stability of DJ-1 WT and DJ-1 phospho-null mutant were compared using cycloheximide chase assay and ubiquitination assay.</P> <P><B>Results</B></P> <P>Here, we showed that DJ-1 directly bound to the catalytic subunit of protein kinase A (PKAcα). We found that PKAcα is responsible for phosphorylation of DJ-1 at the T154 residue. Interestingly, dimerization of DJ-1 was not detected in a DJ-1 T154A mutant. Furthermore, stability of the DJ-1 T154A mutant was dramatically reduced compared with that of wild-type DJ-1. We found that DJ-1 T154A was prone to degradation by the ubiquitin proteasome system (UPS).</P> <P><B>Conclusion</B></P> <P>We identified a novel phosphorylation site of DJ-1. Furthermore, we determined protein kinase A that is responsible for this posttranslational modification. Finally, we demonstrated physiological consequences of this event focusing on dimerization and protein stability of DJ-1.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PKAcα phosphorylates PARK7/DJ1 at the Thr154 residue. </LI> <LI> Disruption of Thr154 phosphorylation is linked to a lower stability. </LI> </UL> </P>