Differential effects of divalent manganese and magnesium on the kinase activity of leucine-rich repeat kinase 2 (LRRK2).
Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson's disease (PD). In this study, we have demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn(2+) in vitro. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn(2+). Compared to 10 mM Mg(2+), 1 mM Mn(2+) reduced ATP K(m) for G2019S from 103 to 1.8 muM and only modestly reduced k(cat) (2.5-fold); as a result, the Mn(2+) increased its k(cat)/K(m) by 22-fold. This change in ATP K(m) was due in large part to an increase in nucleotide affinity. While Mn(2+) also increased ATP affinity and had similar effects on k(cat)/K(m) for LRRK2 WT and R1441C enzymes, it reduced their k(cat) values significantly by 13-17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg(2+) to 10-fold in Mn(2+) at saturating ATP concentrations relative to its K(m). Furthermore, while Mg(2+) yielded optimal V(max) values at Mg(2+) concentration greater than 5 mM, the optimal Mn(2+) concentration for activating LRRK2 catalysis was in the micromolar range with increasing Mn(2+) above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC(50) tested at 100 muM ATP, the apparent K(i) values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg(2+)- and Mn(2+)-mediated reactions except AMP-CPP, an ATP analogue.
Lovitt
Brian
B
Biochemical Pharmacology and Early Leads, Genentech, Inc.,One DNA Way, South San Francisco, California 90480, USA.
Vanderporten
Erica C
EC
Sheng
Zejuan
Z
Zhu
Haitao
H
Drummond
Jake
J
Liu
Yichin
Y
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D009154:Mutation; D004676:Myelin Basic Protein / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics
D002413:Cations, Divalent; D004676:Myelin Basic Protein; D009842:Oligopeptides; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; D008274:Magnesium