Autophosphorylation in the Leucine-Rich Repeat Kinase 2 (LRRK2) GTPase Domain Modifies Kinase and GTP-Binding Activities

Abstract

The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (∼ 7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.

Introduction

The most common known cause of inherited late-onset Parkinson disease (PD) is dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene.1, 2 The 2527-amino-acid LRRK2 protein has both a Ras-like GTP-binding domain and a serine/threonine kinase domain, encoded in a ROC (Ras of complex) guanosine triphosphatase (GTPase) domain and an MLK (mixed-lineage kinase)-like kinase domain.³ Most PD-causative LRRK2 mutations localize to these domains, indicating a critical role for LRRK2 enzymatic activities in disease susceptibility, at least in PD cases with LRRK2 mutations.⁴ LRRK2 is a cytosolic and membrane-associated protein that is expressed in many mammalian tissues and cell types.5, 6, 7 As yet, the endogenous function of the evolutionarily conserved LRRK2 protein kinase is not clear, with putative functions in regulating protein translation, cytoskeleton architecture, and signaling stress responses in the mitogen-activated protein kinase pathway.8, 9, 10, 11, 12, 13, 14

Biochemical changes imparted by LRRK2 mutations may reveal molecular aspects of neurodegeneration in PD and therapeutic targets. Increased in vitro kinase activity due to the most prevalent known LRRK2 pathogenic mutation, G2019S, compared to wild-type (WT) activity, is universally observed under a variety of experimental conditions.3, 15, 16, 17, 18 However, the effects of other mutations on LRRK2 kinase activity, especially those outside of the kinase domain (e.g., the GTPase domain), have not been ubiquitously linked to kinase up-regulation in every study. LRRK2 kinase regulation likely involves a complex mechanism involving intrinsic factors (e.g., the GTPase domain) and extrinsic interacting proteins. For example, GTPase proteins usually require cofactors for enzymatic activity, and these as yet unidentified LRRK2-interacting proteins may be differentially expressed under different experimental conditions. Further delineation of the mechanics of LRRK2 enzymatic activation and downstream function are required to identify pathogenic-disease-associated functions and intervening strategies.

An important clue in resolving the mechanism of LRRK2 enzymatic activation (and thus action of PD-associated mutations) may be the recently described autophosphorylation occurring in residues in or nearby the GTPase domain. Protein kinases commonly autophosphorylate, and in some cases, autophosphorylation is required for initiation of substrate phosphorylation. Conversely, kinase inhibition can result from autophosphorylation, for example, the DAPK (death-associated protein kinase) protein where autophosphorylation of the Ca²⁺ binding domain results in shutdown.¹⁹ Past in vitro mass spectrometry (MS) studies map LRRK2 autophosphorylation sites to its own GTPase domain, suggesting possible kinase control over GTPase function.^20–22 However, mutations within the GTP-binding pocket are known to potently disrupt kinase activity.3, 23, 24 The effects of LRRK2 autophosphorylation on overall activity, initial activation, sustained kinase activity, and GTP-binding activity, are not clear.

PD-causative LRRK2 mutations may plausibly alter specificity of substrate interaction in a true gain-of-function mechanism. Without a bona fide kinase substrate, multiple LRRK2 studies have utilized myelin basic protein (MBP) as a surrogate kinase substrate.18, 23, 25 Several soluble serine/threonine protein kinases can phosphorylate MBP in vitro on multiple sites, and LRRK2 can phosphorylate MBP.3, 18, 23, 26, 27, 28, 29, 30 The sites of LRRK2 phosphorylation on MBP or whether specificity is altered by the PD-associated mutation G2019S has not been previously described. Methods identifying LRRK2-mediated phosphorylation sites on MBP may provide insight into possible gain-of-function mechanisms in LRRK2 PD-associated mutations and help resolve preferred motifs of phosphorylation.

This study further explores the functional effects of LRRK2 autophosphorylation, the relationship between GTP-binding and kinase activities, and gain-of-function mechanisms on phosphorylation site selectivity. We find that the previously tentative link between the GTPase domain and kinase activity, defined largely through mutations that greatly disrupt nucleotide binding in the GTPase domain (i.e., K1347A and T1348N), is further solidified in this study through evaluation of PD-linked mutations and modification of autophosphorylation sites. Analysis of autophosphorylated LRRK2 protein indicates a primary autophosphorylation residue at T1503 that is required for normal kinase activity and GTP binding. We map the sites of LRRK2 phosphorylation on MBP to determine possible alterations in kinase specificity in PD-associated mutants and find no differences except for the rate of activity. We find no evidence that autophosphorylation results in kinase inactivation. On the contrary, we suggest that LRRK2 autophosphorylation serves to propagate protein kinase activity via control of the GTPase domain.