Abstract
BackgroundRecent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.Methodology/Principal FindingsUsing affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2.Conclusions/SignificanceLRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.
Highlights
Parkinson’s disease (PD) is a major human neurodegenerative disease clinically characterized by the motor function deficits as well as non-motor impairment
The results reveal 3 serine phosphorylation sites (S910, 935 and 973) from tryptic peptides and 1 serine phosphorylation site (S912) in chymotryptic peptides of Leucine Rich Repeat Kinase 2 (LRRK2), respectively (Figure 1A) (Figures S1, S2, S3 and S4)
Our study identifies serial molecular events related to the regulation of LRRK2: multiple novel phosphorylation sites (S910, S912, S935 and S973, all in the N-terminal region), potential phosphorylation of S935 by protein kinase A (PKA), and phosphorylated S935 (pS935)-dependent 14-3-3 binding of LRRK2
Summary
Parkinson’s disease (PD) is a major human neurodegenerative disease clinically characterized by the motor function deficits as well as non-motor impairment. The pathogenic mechanism of PD remains largely undefined. The recent identification of genetic mutations that are associated with familial PD provides an entry to uncover cellular and molecular pathways that lead to the disease. Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) have been linked to the most common familial form and some sporadic forms of PD [1,2]. Because many LRRK2 mutation carriers exhibit typical PD symptoms indistinguishable from idiopathic PD cases, it is hypothesized that an understanding of the biology and pathophysiology of LRRK2 will provide new opportunities to develop effective treatments of PD. Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson’s disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
