Enzymatic activity of LRRK2

LRRK2 harbors both a GTPase and kinase domain, an extremely rare arrangement found only in LRRK1 and possibly the DAPK1 protein in mammals. A solid precedent for
GTPase activity dependent modification of protein kinase activity suggests that the enzymatic domains encoded inLRRK2 may function as a self-regulatory apparatus. The most common LRRK2 mutations localize to the kinase domain (amino acid G2019) and the GTPase domain (amino acid R1441), implicating enzymatic output as critical to PD. Beyond a domain-prediction analysis that helps define conserved features and potentially critical residues, in silico approaches do not further predict LRRK2 function in cells. The LRRK2 kinase domain possesses highest sequence homology
to the mixed-lineage kinase (MLK) subfamily of MAPKKK proteins but differs in the critical amino acids that define MLK proteins, whereas the GTPase domain displays
distinct architecture reminiscent of Rab-like GTPases. As one of the largest protein kinases in the mammalian kinome, LRRK2 will necessarily resist characterization by the usual gauntlet of biochemical assays that have historically well served the characterization of other protein kinases. Nevertheless, tremendous advances in a short time have outlined a surprising consensus story for the impact of PD causing
mutations on protein function. Before a functional description of the LRRK2 protein, a functional description of LRRK1 suggested that intrinsic kinase activity was stimulated on GTPase activation. This phenomenon holds true for LRRK2 as well, in which application of GTP or GTPgS spurs modest increases in kinase output. However, a clear relation between kinase activity and GTPase activity is defined through the us of specific mutations that ablate either GTPase activity, kinase activity, or both activities. Mutations in the conserved ATP binding pocket site, in the consensus residues of the activation loop, or mutation within the proton-acceptor site have no apparent effect on GTP binding. In contrast, mutations in the
GTPase domain thoroughly ablate kinase activity. Thus, LRRK2 functions as a signal-transduction pathway encoded into a single protein. The activity of LRRK2 is
perhaps an ancient design, because the orthologue GbpC in slime mold operates in a similar manner, although GbpC also encodes GEF domains upstream of GTPase activity.
Through functional descriptions of GbpC, LRRK1, and LRRK2 protein, the encoded domains function in a signal F2c cascade ultimately to regulate kinase output.In an initial functional description of the LRRK2 protein, the most common PD-associated mutations, G2019S and R1441C, enhanced kinase activity but failed to alter other
basic biochemical properties, such as protein localization and turnover. PD-causing mutations in the GTPase domain likewise enhance the proportion of GTP-bound protein in pull-down experiments, whereas mutations in the kinase domain have no effect on the proportion of GTP-bound protein. Additional studies demonstrate that PD-causing
mutations in the GTPase domain do not result in a higher affinity for GTP; rather, GTP hydrolysis is disrupted because of PD-associated mutations, thereby prolonging GTP-bound states and an activated GTPase domain. Missense mutations in the LRRK2 gene that segregate with disease in families and are therefore likely pathogenic variants all result in enhanced kinase activity in vitro, although not all
laboratories demonstrate a significant difference between kinase activity associated with wild-type protein and that containing PD-causing mutations. Differences in assay
protocols and the lack of a relevant substrate and robust kinase-dependent phenotype in cells combine to prevent the assumption that kinase output is the true and only possible functional link between LRRK2 protein and PD. However, the available data, when taken as a whole, suggest that kinase activity represents the final output of activity responsible for pathogenesis.