A method for identifying a compound expected to be useful in modulating a LRRK2 protein kinase activity, the method comprising the steps of (1) determining whether a test compound modulates the protein kinase activity of a LRRK2 polypeptide on a substrate Ezrin/Radixin/moesin (ERM) family polypeptide and (2) selecting a compound which modulates the LRRK2 polypeptide protein kinase activity. Such a compound may be useful in treating Parkinson's Disease or Parkinsonism. A catalytically active fragment of LRRK2 is identified, requiring the GTPase, COR and kinase domains as well as the WD_40-like motif and C-terminal tail.
1. A method for identifying a compound expected to be useful in inhibiting LRRK2 protein kinase activity, the method comprising the steps of:
(1) determining whether a test compound inhibits the protein kinase activity of a LRRK2 polypeptide on a substrate polypeptide, and
(2) selecting a compound which inhibits said LRRK2 polypeptide protein kinase activity,
wherein the substrate polypeptide comprises a sequence selected from the group consisting of RLGRDKYKTLRQIRQ (SEQ ID NO:1), RLGRDKYKSLRQIRQ (SEQ ID NO:3), WWRFYTLRRA (SEQ ID NO:65), WWKFYTLRRA (SEQ ID NO:47), WWRFYTLRKA (SEQ ID NO:48), MRQGRDKYKTLRQIRQGNTKQRIDEFEAL (SEQ ID NO:44), VKAGRDKYKTLRQIRQGNTKQRIDEFEAM (SEQ ID NO:45), RLGRDKYK(T/S)LRQIRQGNTKQR (SEQ ID NO:4), RLGWWRFYTLRRARQGNTKQR (SEQ ID NO:51), RLGWWKFYTLRRARQGNTKQR (SEQ ID NO:49), RLGWWRFYTLRKARQGNTKQR (SEQ ID NO:50), moesin (SEQ ID NO:39), ezrin (SEQ ID NO:70), radixin (SEQ ID NO:41), or a fusion peptide thereof.
2. The method of claim 1 wherein the LRRK2 polypeptide is wild type human LRRK2 (SEQ ID NO:69); or a fragment comprising at least residues 1326-2527 of wild type human LRRK2; or a fusion either thereof.
3. The method of claim 1 wherein the LRRK2 polypeptide is human LRRK2 having a naturally occurring mutation of wild type human LRRK2; or a fragment corresponding to at least residues 1326-2527 of human LRRK2; or a fusion either thereof.
4. The method of claim 3 wherein the naturally occurring mutation of human LRRK2 is a mutation associated with Parkinson's Disease (PD).
5. The method of claim 3 wherein the mutation, using the numbering of wild type human LRRK2, is G2019S.
6. The method of claim 2 wherein the LRRK2 polypeptide is a GST fusion polypeptide.
7. The method of claim 5 wherein the LRRK2 polypeptide is GST-LRRK2[1326-2527, G2019S].
8. The method of claim 1 wherein the substrate polypeptide is from 10 to 100 amino acids, is selected from the group consisting of RLGRDKYKTLRQIRQ (SEQ ID NO:1), RLGRDKYKSLRQIRQ (SEQ ID NO:3), WWRFYTLRRA (SEQ ID NO:65), WWKFYTLRRA (SEQ ID NO:47), WWRFYTLRKA (SEQ ID NO:48), MRQGRDKYKTLRQIRQGNTKQRIDEFEAL (SEQ ID NO:44), VKAGRDKYKTLRQIRQGNTKQRIDEFEAM (SEQ ID NO:45), RLGRDKYK(T/S)LRQIRQGNTKQR (SEQ ID NO:4), RLGWWRFYTLRRARQGNTKQR (SEQ ID NO:51), RLGWWKFYTLRRARQGNTKQR (SEQ ID NO:49), RLGWWRFYTLRKARQGNTKQR (SEQ ID NO:50), moesin (SEQ ID NO:39), ezrin (SEQ ID NO:70), radixin (SEQ ID NO:41), or a fusion peptide thereof, and optionally includes up to ten conservative or non-conservative amino acid substitutions at residues other than the T/S residue.