LRRK2 is one of the largest kinases in the human kinome, with less than ten kinases (mostly predicted kinases) derived from the PIKK family, the Trio family and the MLCK family encoding proteins larger than LRRK2, the majority of which remain uncharacterized on a molecular level. While all large kinase proteins display unique configurations of conserved protein domains, LRRK2 and the related LRRK1 gene encode the only proteins within the mammalian genome that include both kinase and GTPase domains, with the possible exception of the calcium-calmodulin-dependent death-associated protein kinase 1 (DAPK1) that contains a possible GTPase-like domain [6
]. Since the most common mutations linking LRRK2 with susceptibility to PD occur within the LRRK2 kinase and GTPase domains, attention and intense speculation immediately revolved around the enzymatic output of LRRK2 protein prior to a functional description of the protein.
As a highly evolutionarily conserved kinase-protein arguably present in even some single-celled organisms with broad expression throughout mammalian development, LRRK2 protein plausibly performs dozens of functions in dozens of cells types. Perhaps the best way to study LRRK2 as a cause and potential therapeutic target for PD revolves around understanding how PD-associated mutant LRRK2 protein deviates from normal protein. The first functional description of LRRK2 protein included a comparison of the most common mutation found in PD cases, G2019S, which localizes to the activation loop kinase sub-domain, together with normal LRRK2 protein sequence as found in unaffected control brain tissue [22
]. While the basic biochemical properties of wild-type and mutant protein such as protein localization and stability are comparable, at least in transiently transfected cell lines, the influence of the PD-linked mutations is clear in experiments designed to assess enzymatic activity. The G2019S mutation enhances kinase activity in autophosphorylation assays and phosphorylation of a generic kinase substrate myelin basic protein [22
]. These findings, derived from in vitro
based assays, complement genetic studies that suggest dominant inheritance and likely gain of function pathogenic mechanisms.
LRRK2 belongs to the tyrosine kinase-like protein family in humans [40
]. Despite the nomenclature, all described kinase proteins within the tyrosine kinase-like family display serine and/or threonine activity without examples of tyrosine kinase activity. Separation of internal LRRK2 residues labeled during autophosphorylation reveal both serine and threonine kinase activity, typical for tyrosine kinase like proteins [41
]. Although the LRRK2 kinase domain displays highest sequence homology to the mixed-lineage kinase (MLK) subfamily of MAPKKK protein kinases, so named due to kinase sub-domain structure resembling both tyrosine and serine/threonine kinases, LRRK2 differs from the consensus critical amino acids that define mixed-lineage kinases [41
]. Thus, LRRK1 and LRRK2 protein kinase domains have unique sub-domain characteristics that clearly delineate from MLKs and other MAPKKK proteins. However, like MLKs, LRRK2 forms protein dimers in cells and dimerization is dependent on kinase activity [42
]. The LRRK2 kinase domain deviates from nearly all protein kinases from the consensus DFG…APE activation loop motif [43
]. Since the most common LRRK2 mutation G2019S alters the unusual DYG…APE motif to DYS…APE, this unique region seems critical to the normal regulation of kinase activity, although the serine and threonine residues within the activation loop have not been formally shown as autophosphorylation and possible auto-activation sites. Thus, the high divergence of the LRRK2 kinase domain from other well known protein kinases may provide additional opportunities for specificity in therapeutic approaches but prevents comparisons to homologous kinases for clues to functionality.
Since the first description of LRRK2 kinase activity, additional studies that involve immunoprecipitation of over-expressed LRRK2 protein and assessment of autophosphorylation activity in vitro
demonstrate that pathogenic PD-linked mutations increase apparent kinase activity [41
]. Protein fragments containing the LRRK2 kinase domain isolated from E. coli
likewise demonstrate auto-phosphorylation activity and kinase activity with MBP as substrate, and the most common LRRK2 mutation, G2019S, imparts a significant increase in kinase activity [44
]. Notably, kinase fragments derived from the E. coli
system are guaranteed devoid of contaminating endogenous serine/threonine and tyrosine kinases. The pathogenic LRRK2 mutation I2020T had no significant effect on activity versus wild-type protein, in contrast with experiments in mammalian systems that demonstrate either increases or decreases in activity [41
]. While the most common LRRK2 mutation G2019S faithfully reproduces enhanced autophosphorylation, other pathogenic LRRK2 mutations fail to reproduce enhanced activity uniformly among different laboratories. G2019S, as opposed to other mutations, may provide an additional autophosphorylation site in the activation loop in addition to enhancing auto-activation that would increase autophosphorylation signal even when a particular assay exceeds linearity. Differences in kinase reaction protocol, contaminating but highly active serine/threonine kinases in kinase reactions, lack of linearity of autophosphorylation assays, lack of a bone fide kinase substrate and sequence differences among different LRRK2 clones and cell lines used for recombinant protein production in different laboratories all likely contribute to variation. Fortunately, the unresolved in vitro
kinase story does not end with the LRRK2 kinase domain since a second enzymatic domain encoded in the LRRK2 protein, the GTPase domain, provides an additional opportunity to understand the effects of PD-linked LRRK2 mutations on LRRK2 protein function and LRRK2 kinase regulation.
The first descriptions of LRRK2 GTPase activity utilized measurements of GTP-bound LRRK2 protein in cell lysates derived from transiently transfected cells [41
]. These estimations revealed that pathogenic PD mutations near the GTPase domain increased the proportion of LRRK2 bound to GTP, while mutations near the kinase domain had no significant effect on the proportion of GTP bound LRRK2. Beyond GTP-bound LRRK2 as a surrogate measure of GTPase activity, more in depth studies reveal that the most common LRRK2 mutations in the LRRK2 GTPase domain, R1441C and R1441G, do not directly enhance GTP binding but convincingly decrease GTP-hydrolysis activity [47
]. Structural studies demonstrate that the R1441 residue is located at the interface of two GTPase monomer structures and that the mutation likely diminishes GTPase activity from a loss of stabilization of the GTPase dimer [50
]. Taken together, the data suggest that the pathogenic PD mutations localized to the GTPase domain prolong the binding to GTP compared to normal LRRK2 protein.
Concurrent with data delineating the effect of PD-associated mutations on GTPase activity, multiple studies report that artificial mutations within the GTPase domain that ablate GTPase activity completely inhibit kinase activity, whereas mutations that ablate kinase activity have no effect on GTPase activity [41
]. Thus, the LRRK2 protein encodes a self-regulatory module in the GTPase domain that controls kinase output, where mutations that affect GTPase activity necessarily affect kinase activity. This model explains how mutations over 500 amino acid residues away from the kinase domain affect kinase activity, and suggest that pathogenic LRRK2 mutations may influence kinase function either through direct alteration of the kinase domain, alteration of GTPase activity or potentially through altering the interaction of the GTPase and kinase domain. The pathogenic Y1699C mutation localized between the GTPase and kinase domains may alter the structure of LRRK2 protein and influence the contact between the GTPase and kinase domain, although this speculation awaits structural studies with full length recombinant LRRK2 protein.
encodes a protein comprised of elements usually split into two, three or more distinct proteins to form a signal transduction pathway. Descriptions of distant mammalian LRRK relatives in dictyostelium likewise confirm that multiple regulatory domains function in a serial fashion to modify kinase output [52
]. In the GbpC protein, a RasGEF domain enhances GTPase activity which in turn is required for kinase activity. Thus, a signal transduction pathway is encoded into a single protein, with opportunities at each step for additional regulation. Perhaps the encoded domains in LRRK2 and related proteins represent such specialized and exact components in controlling kinase output that splitting up the domains into separate proteins would yield insufficient control over kinase output. Regardless of the reason for the unique domains structure, evolution has clearly selected for the arrangement that seemingly positions kinase output as the critical and defining feature of the proteins.