Fig. 4.

LRRK2 kinase regulation by ROC. (A) Stereo model of the LRRK2 functional GTPase unit (yellow and green for each monomer) superimposed on Ras (PDB ID 1ctq, red). Two key residues important in the regulation of Ras activity after GTP binding are Q61 and G12 (shown in red, labeled in red); the equivalent residues in LRRK2 are R1398 and T1343, respectively (shown in yellow, labeled in black). (B) Ras-like mutations R1398Q and T1343G were made individually and together (TQ/RG, lane 5) in the full-length LRRK2 construct, and their effects on autophosphorylation of the kinase were tested. (Upper) Autoradiogram with ³²P. (Lower) A blot for LRRK2. Quantification (n = 3, graph) shows that although each Ras-like mutation alone has no effect on kinase activity, the double TG/RQ caused a decrease in net kinase activity. **, P < 0.01; ***, P < 0.001 by ANOVA with Bonferroni's multiple comparison test. (C) ROC domain has intrinsic association with COR domain. Truncated domain proteins used for interaction assay are as follows: ROC–COR–kinase protein (lane 1), COR domain only (lane 2), and kinase domain only (lane 3), respectively, blotted with a myc tag. Their corresponding pull-down results with ROC domain as a GST fusion protein are shown in lanes 4–6, respectively. GST was used as negative control in lanes 7–9. (D) Model for ROC regulation of kinase activity. Dimeric ROC GTPase (yellow and green ovals) act as binary switches in regulation of kinase activation. Upon binding of GTP (stars), the activated ROC induces dimerization of the COR domain (barrels). The dimerization of COR domain further induces self-association of kinase domain, resulting in its autophosphorylation and activation (red ovals). Through hydrolysis of GTP to GDP (black oval), conformational changes in ROC disrupt the dimeric association of COR and kinase domain, inactivating the kinase (blue ovals). GTPase activating protein (GAP) and guanine nucleotide exchange factor (GEF) are not known.