A continuous and direct assay to monitor leucine-rich repeat kinase 2 activity.
Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain enzyme displaying activities of GTP hydrolase and protein threonine/serine kinase in separate domains. Mutations in both catalytic domains have been linked to the onset of Parkinson's disease, which triggered high interest in this enzyme as a potential target for drug development, particularly focusing on inhibition of the kinase activity. However, available activity assays are discontinuous, involving either radioactivity detection or coupling with antibodies. Here we describe a continuous and direct assay for LRRK2 kinase activity, combining a reported peptide sequence optimized for LRRK2 binding and an established strategy for fluorescence emission on magnesium ion chelation by phosphorylated peptides carrying an artificial amino acid. The assay was employed to evaluate apparent steady-state parameters for the wild type and two mutant forms of LRRK2 associated with Parkinson's disease as well as to probe the effects of GTP, GDP, and autophosphorylation on the kinase activity of the enzyme. Staurosporine was evaluated as an inhibitor of the wild-type enzyme. It is expected that this assay will aid in mechanistic investigations of LRRK2.
Structural Biology and Biophysics Group, Center for Chemistry Innovation and Excellence, Pfizer Worldwide Medicinal Chemistry, Groton, CT 06340, USA. Electronic address: rafaelguimaraes.dasilva@pfizer.com.
Rafael G
RG
Silva
Structural Biology and Biophysics Group, Center for Chemistry Innovation and Excellence, Pfizer Worldwide Medicinal Chemistry, Groton, CT 06340, USA.
Kieran F
KF
Geoghegan
Structural Biology and Biophysics Group, Center for Chemistry Innovation and Excellence, Pfizer Worldwide Medicinal Chemistry, Groton, CT 06340, USA.
Xiayang
X
Qiu
Structural Biology and Biophysics Group, Center for Chemistry Innovation and Excellence, Pfizer Worldwide Medicinal Chemistry, Groton, CT 06340, USA.
Ann
A
Aulabaugh
D000595:Amino Acid Sequence; D057075:Enzyme Assays / Q000379:methods*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019311:Staurosporine / Q000494:pharmacology
D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine
Chelation-enhanced fluorescence; LRRK2 kinetics; LRRK2 mutations; Leucine-rich repeat kinase 2; Parkinson’s disease