The PD (Parkinson's disease) protein LRRK2 (leucine-rich repeat kinase 2) occurs in cells as a highly phosphorylated protein, with the majority of phosphosites clustering in the region between the ankyrin repeat and leucine-rich repeat domains. The observation that several pathogenic variants of LRRK2 display strongly reduced cellular phosphorylation suggests that phosphorylation of LRRK2 is involved in the PD pathological process. Furthermore, treatment of cells with inhibitors of LRRK2 kinase activity, which are currently considered as potential disease-modifying therapeutics for PD, leads to a rapid decrease in the phosphorylation levels of LRRK2. For these reasons, understanding the cellular role and regulation of LRRK2 as a kinase and as a substrate has become the focus of intense investigation. In the present review, we discuss what is currently known about the cellular phosphorylation of LRRK2 and how this relates to its function and dysfunction.

Introduction

In 2004, mutations in LRRK2 (leucine-rich repeat kinase 2) were identified as the cause of PARK8-linked PD (Parkinson's disease) [1,2]. LRRK2 is a large multidomain protein with both GTPase and kinase functions [3]. Although much attention has focused on the study of the interplay between both enzymatic functions and their role in pathology (reviewed in [4,5]), another major feature of LRRK2 which has recently come to light is that LRRK2 is a highly phosphorylated protein [6]. An important consideration in discussing LRRK2 phosphorylation is the distinction between autophosphorylation and cellular phosphorylation events. Autophosphorylation of LRRK2 is observed when purified LRRK2 is incubated in vitro with ATP and occurs at multiple sites, the majority of which have been mapped to the ROC (Ras of complex proteins) GTPase domain [68] (Table 1 and Figure 1). Yet the output of LRRK2 kinase activity in the cell is poorly understood, since neither LRRK2 autophosphorylation nor direct LRRK2-mediated phosphorylation of candidate substrates has been confirmed in mammalian cells. Although phosphorylation at LRRK2 autophosphorylation sites is very rarely observed in cells under basal cellular conditions, LRRK2 is phosphorylated in the cell at multiple sites between the ankyrin and LRR (leucine-rich repeat) domains [6]. Intriguingly, this cellular constitutive LRRK2 phosphorylation is strongly reduced in the case of several pathogenic LRRK2 mutations as well as after pharmacological LRRK2 kinase inhibition [912]. Furthermore, the K1906M kinase-dead variant of LRRK2 does not affect cellular LRRK2 phosphorylation levels, pointing to a complex and indirect role for LRRK2 in regulating its own cellular phosphorylation (Table 2). Therefore more insight into the regulation of cellular LRRK2 phosphorylation would be of great value to better understand (i) the implications of the LRRK2 phosphorylation level for its function and dysfunction, and (ii) the cellular consequences of LRRK2 kinase inhibition, which has been proposed as a potential PD therapeutic strategy (reviewed in [13]). In the present review, we focus on the different aspects of cellular LRRK2 phosphorylation. We discuss LRRK2 as a kinase and as substrate, the potential interplay between both as well as the effect of mutations.

Table 1
Overview of reported LRRK2 phosphorylation sites

List of LRRK2 phosphorylation sites that could be determined with certainty; sites reported at least twice are depicted in bold. ANK, ankyrin; ARM, armadillo; COR, C-terminal of ROC; FL, full-length; GS, G2019S; HEK, human embryonic kidney; KIN, kinase; P-Ab, phospho-specific antibody.

Amino acid Domain LRRK2 protein Conditions Method 
Thr424 ARM WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr524 ARM WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr776 ANK WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr826 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr833 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr838 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Ser860 Between ANK and LRR WT, FL, endogenous Swiss 3T3 cells and overexpression in HEK-293T cells [12Cellular phosphosites MS 
Ser910 Between ANK and LRR WT, FL, endogenous Swiss 3T3 cells [1212,], and overexpression in mouse brain [3737,] and HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser912 Between ANK and LRR WT, FL, overexpression in mouse brain [37Cellular phosphosite MS 
Ser926 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6Cellular phosphosite MS 
Ser935 Between ANK and LRR WT, FL, endogenous Swiss 3T3 cells [1212,], and overexpression in mouse brain [3737,] and HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser955 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser973 Between ANK and LRR WT, FL, overexpression in mouse brain [3737,] and HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser976 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [12Cellular phosphosite MS 
Ser1124 LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Ser1253 LRR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1283 LRR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1292 LRR WT [66,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation MS 
Tyr1332 Between LRR and ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1343 ROC WT [4444,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation MS 
Ser1345 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1348 ROC WT, FL, overexpression in HEK-293T cells [44In vitro autophosphorylation MS 
Thr1357 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1368 ROC WT [4444,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation MS 
Ser1403 ROC GS, Δ1326–2527 [88,] and FL [2828,], overexpression in Sf9 [88,] and HEK-293T [2828 ] cells In vitro autophosphorylation MS 
Thr1404 ROC GS, Δ1326–2527 [88,] and FL [2828,], overexpression in Sf9 [88,] and HEK-293T [2828 ] cells In vitro autophosphorylation MS 
Thr1410 ROC WT [66,77,2828,] and GS [88,2828,], Δ970–2527 [77,], Δ1326–2527 [88,] and FL [66,2828,], overexpression in Sf9 [66,77,] and HEK-293T [66,2828 ] cells In vitro autophosphorylation, and cellular phosphosite [28MS [66,88,2828,], P-Ab [2828 ] 
Thr1452 ROC WT [66,77,] and GS [2828,], Δ970–25271 and FL [66,2828,], overexpression in Sf91 and HEK-293T [66,2828 ] cells In vitro autophosphorylation MS 
Ser1457 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1467 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1491 ROC WT [66,88,4444,], GS [2828,], ROC [77,], Δ1326–2527 [88,] and FL [66,2828,4444,], overexpression in Escherichia coli [77,], Sf9 [88,] and HEK-293T [66,4444 ] cells In vitro phosphorylation with LRRK2Δ970–2527 [77,] and in vitro autophosphorylation [66,88,2828,4444 ] MS 
Thr1503 ROC WT7,[66,77,4444,], GS [2828,], Δ970–2527 [77,], FL [66,2828,4444,] and ROC [77,], overexpression in Escherichia coli [77,], and overexpression in Sf9 [77,] and HEK-293T [66,2828,4444 ] cells In vitro autophosphorylation [66,77,2828,4444,] and phosphorylation with LRRK2Δ970–2527 [77 ] MS 
Ser1536 COR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1612 COR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1647 COR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1849 Between COR and KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1853 Between COR and KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1912 KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1913 KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1967 KIN Δ1326–2527, overexpression in Sf9 cells [8In vitro autophosphorylation MS 
Thr1969 KIN Δ1326–2527, overexpression in Sf9 cells [8In vitro autophosphorylation MS 
Thr2031 KIN WT [4545,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation P-Ab [4545,], MS [2828 ] 
Thr2032 KIN WT, FL, overexpression in HEK-293T cells [45In vitro autophosphorylation P-Ab 
Thr2035 KIN WT, FL, overexpression in HEK-293T cells [45In vitro autophosphorylation P-Ab 
Ser2257 WD40 GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr2483 WD40 WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr2524 C-terminus GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Amino acid Domain LRRK2 protein Conditions Method 
Thr424 ARM WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr524 ARM WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr776 ANK WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr826 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr833 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr838 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Ser860 Between ANK and LRR WT, FL, endogenous Swiss 3T3 cells and overexpression in HEK-293T cells [12Cellular phosphosites MS 
Ser910 Between ANK and LRR WT, FL, endogenous Swiss 3T3 cells [1212,], and overexpression in mouse brain [3737,] and HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser912 Between ANK and LRR WT, FL, overexpression in mouse brain [37Cellular phosphosite MS 
Ser926 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [6Cellular phosphosite MS 
Ser935 Between ANK and LRR WT, FL, endogenous Swiss 3T3 cells [1212,], and overexpression in mouse brain [3737,] and HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser955 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser973 Between ANK and LRR WT, FL, overexpression in mouse brain [3737,] and HEK-293T cells [1212,2727 ] Cellular phosphosite MS 
Ser976 Between ANK and LRR WT, FL, overexpression in HEK-293T cells [12Cellular phosphosite MS 
Ser1124 LRR WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Ser1253 LRR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1283 LRR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1292 LRR WT [66,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation MS 
Tyr1332 Between LRR and ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1343 ROC WT [4444,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation MS 
Ser1345 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1348 ROC WT, FL, overexpression in HEK-293T cells [44In vitro autophosphorylation MS 
Thr1357 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1368 ROC WT [4444,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation MS 
Ser1403 ROC GS, Δ1326–2527 [88,] and FL [2828,], overexpression in Sf9 [88,] and HEK-293T [2828 ] cells In vitro autophosphorylation MS 
Thr1404 ROC GS, Δ1326–2527 [88,] and FL [2828,], overexpression in Sf9 [88,] and HEK-293T [2828 ] cells In vitro autophosphorylation MS 
Thr1410 ROC WT [66,77,2828,] and GS [88,2828,], Δ970–2527 [77,], Δ1326–2527 [88,] and FL [66,2828,], overexpression in Sf9 [66,77,] and HEK-293T [66,2828 ] cells In vitro autophosphorylation, and cellular phosphosite [28MS [66,88,2828,], P-Ab [2828 ] 
Thr1452 ROC WT [66,77,] and GS [2828,], Δ970–25271 and FL [66,2828,], overexpression in Sf91 and HEK-293T [66,2828 ] cells In vitro autophosphorylation MS 
Ser1457 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1467 ROC GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1491 ROC WT [66,88,4444,], GS [2828,], ROC [77,], Δ1326–2527 [88,] and FL [66,2828,4444,], overexpression in Escherichia coli [77,], Sf9 [88,] and HEK-293T [66,4444 ] cells In vitro phosphorylation with LRRK2Δ970–2527 [77,] and in vitro autophosphorylation [66,88,2828,4444 ] MS 
Thr1503 ROC WT7,[66,77,4444,], GS [2828,], Δ970–2527 [77,], FL [66,2828,4444,] and ROC [77,], overexpression in Escherichia coli [77,], and overexpression in Sf9 [77,] and HEK-293T [66,2828,4444 ] cells In vitro autophosphorylation [66,77,2828,4444,] and phosphorylation with LRRK2Δ970–2527 [77 ] MS 
Ser1536 COR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1612 COR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1647 COR GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1849 Between COR and KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1853 Between COR and KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1912 KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Ser1913 KIN GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr1967 KIN Δ1326–2527, overexpression in Sf9 cells [8In vitro autophosphorylation MS 
Thr1969 KIN Δ1326–2527, overexpression in Sf9 cells [8In vitro autophosphorylation MS 
Thr2031 KIN WT [4545,] and GS [2828 ], FL, overexpression in HEK-293T cells In vitro autophosphorylation P-Ab [4545,], MS [2828 ] 
Thr2032 KIN WT, FL, overexpression in HEK-293T cells [45In vitro autophosphorylation P-Ab 
Thr2035 KIN WT, FL, overexpression in HEK-293T cells [45In vitro autophosphorylation P-Ab 
Ser2257 WD40 GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Thr2483 WD40 WT, FL, overexpression in HEK-293T cells [6In vitro autophosphorylation MS 
Thr2524 C-terminus GS, FL, overexpression in HEK-293T cells [28In vitro autophosphorylation MS 
Table 2
LRRK2 mutations with corresponding kinase and GTPase activity and cellular phosphorylation level after overexpression of full-length protein in HEK-293T cells
Mutation GTPase activity Kinase activity Cellular phosphorylation 
Pathogenic mutations    
 R1441C ↓ [4= [4↓ [10,12
 R1441G ↓ [4= [4↓↓ [10,12,37
 R1441H ↓ [12
 Y1699C ↓ [4= [4↓↓ [10,12,37
 G2019S = [35↑ [4= [10,12,37], ↓* [37
 I2020T = [35= [4↓ ↓ [10,12
Functional mutations    
 GTP-binding-deficient ↓ [27,32,33,36,39↓ [16,27,32,33,39,46↓ [39,40
 Kinase-dead = [39↓ [27,39= [8,10,12,27,37,39], ↓† [28
Mutation GTPase activity Kinase activity Cellular phosphorylation 
Pathogenic mutations    
 R1441C ↓ [4= [4↓ [10,12
 R1441G ↓ [4= [4↓↓ [10,12,37
 R1441H ↓ [12
 Y1699C ↓ [4= [4↓↓ [10,12,37
 G2019S = [35↑ [4= [10,12,37], ↓* [37
 I2020T = [35= [4↓ ↓ [10,12
Functional mutations    
 GTP-binding-deficient ↓ [27,32,33,36,39↓ [16,27,32,33,39,46↓ [39,40
 Kinase-dead = [39↓ [27,39= [8,10,12,27,37,39], ↓† [28
*

Observed in G2019S LRRK2 from BAC transgenic mice.

Measured only at Thr1410. GTP-binding-deficient mutants are T1348N or K1347A, kinase-dead mutants are K1906M, D1994N or D2017A.

Schematic representation of LRRK2

Figure 1
Schematic representation of LRRK2

Pathogenic mutations are depicted in red on top and phosphorylation sites that are reported at least twice are shown below. In vitro autophosphorylation sites are depicted in green, and cellular phosphorylation sites are in blue.

Figure 1
Schematic representation of LRRK2

Pathogenic mutations are depicted in red on top and phosphorylation sites that are reported at least twice are shown below. In vitro autophosphorylation sites are depicted in green, and cellular phosphorylation sites are in blue.

Cellular LRRK2 phosphorylation

When LRRK2 was identified as the cause of PARK8-linked PD, structural homology revealed the presence of a kinase domain [2]. Autophosphorylation assays as well as in vitro phosphorylation of generic substrates confirmed kinase activity of overexpressed [1518] as well as endogenous [19] LRRK2. To date, several proteins have been proposed as LRRK2 kinase substrates based on in vitro data or indirect cellular evidence (reviewed in [20]). However, evidence that these proteins are true physiological LRRK2 substrates is still poor. This, together with strong in vitro autophosphorylation activity, raised the idea that autophosphorylation is the main LRRK2 kinase output or can at least be used as a surrogate measure of kinase activity. Since kinase activity is typically regulated by phosphorylation [2123] and the most common pathogenic mutation G2019S shows enhanced kinase activity [4], in vitro LRRK2 autophosphorylation sites were deter-mined in order to gain more insight into the (auto)regulation and consequences of LRRK2 autophosphorylation [68,24] (Table 1 and Figure 1). These studies revealed that, although autophosphorylation sites are found over the whole protein, they preferentially cluster in its GTPase domain [6,7].

Interestingly, Berger et al. [25] reported that membrane-associated LRRK2 displays a reduced cellular phosphorylation level, but increased in vitro autophosphorylation activity compared with cytosolic LRRK2, suggesting that LRRK2 autophosphorylation and cellular LRRK2 phosphorylation are regulated by distinct mechanisms involving different, as yet unknown, players. Indeed, MS as well as metabolic labelling experiments revealed that WT (wild-type) LRRK2 is phosphorylated in the cell to the same extent as a kinase-dead variant, pointing to phosphorylation of LRRK2 by other kinases [26,27]. Using a phospho-Thr1410-specific antibody, Pungaliya et al. [28] reported cellular phosphorylation of this autophosphorylation site in WT LRRK2, but not in the kinase-dead mutant. Interestingly, using truncated LRRK2 lacking the N-terminal segment (ΔLRRK21326–2527), Kamikawaji et al. [8] reported via metabolic labelling phosphorylation of the WT form, but not of the kinase-dead mutant, suggesting cellular autophosphorylation of this truncated LRRK2 fragment. To explore further the role of LRRK2 (auto)phosphorylation, Gloeckner et al. [6] performed a profound phosphopeptide analysis. Strikingly, none of the 23 identified LRRK2 autophosphorylation sites was found to be phosphorylated in the cell. Rather, all cellular phosphorylation sites are serine residues and cluster in a narrow region N-terminal of the LRR domain [6].

Next, the finding that 14-3-3 proteins can bind LRRK2 at some of the constitutive LRRK2 phosphorylation sites in a phospho-dependent way provided more insight into the importance and regulation of these sites [12]. Indeed, phosphorylation of Ser910 and Ser935 is necessary for 14-3-3 binding and alanine substitutions at these sites disrupt the binding. A common feature of 14-3-3 proteins is to bind to partner proteins and alter their subcellular localization [29]. Binding of 14-3-3 leads to a uniform LRRK2 distribution throughout the cytoplasm; however, LRRK2 with reduced cellular phosphorylation at Ser910 and Ser935 and hence impaired 14-3-3 binding, accumulates in cytoplasmic pools [12]. 14-3-3 proteins seem to bind specifically to Ser910 and Ser935, and not to (phosphorylated) Ser955 and Ser973. Consequently, alanine mutations at these sites do not affect 14-3-3 binding and do not lead to LRRK2 relocalization [10,12]. Interestingly, the finding that an alanine substitution at Ser910 and Ser935 leads to dephosphorylation of Ser973 suggests that phosphorylation of Ser973 is, at least partly, dependent on phosphorylation of other sites [10]. Yet, since alanine substitutions at Ser955 and Ser973 do not affect 14-3-3 binding at phospho-Ser910 and phospho-Ser935, the latter sites do not seem to depend on phosphorylation at Ser955 and Ser973.

Since pathogenic LRRK2 mutations are clustered in the so-called catalytic core (Figure 1), much effort has been devoted to investigate whether these mutations affect LRRK2 enzymatic function [30,31]. A meta-analysis of in vitro LRRK2 autophosphorylation intensity shows that for the most common pathogenic mutation, G2019S, a 2–3-fold enhanced kinase activity compared with WT is observed, whereas other prevalent mutants (R1441C/G, Y1699C and I2020T) on average display activities comparable with that of WT [4]. On the other hand, decreased GTPase activity was shown for mutations R1441G/C and Y1699C [3236]. Recently, an extensive comparison between 41 LRRK2 variants revealed that five out of the six confirmed pathogenic mutants (R1441C/G/H, Y1699C and I2020T), display strongly reduced cellular phosphorylation with impaired 14-3-3 binding and consequent LRRK2 accumulation in cytoplasmic pools [10,12], suggesting a role for disturbed cellular phosphorylation in LRRK2 pathogenesis. Intriguingly, the cellular phosphorylation level of LRRK2 G2019S is comparable with that of WT LRRK2 [10,12,37]. Also, LRRK2 from lymphoblastoid cells derived from a PD patient harbouring a homozygous G2019S mutation showed a similar phosphorylation level at Ser910 and Ser935 compared with WT LRRK2 derived from a healthy control [11]. Similarly, Li et al. [37] reported that G2019S LRRK2 isolated from BAC (bacterial artificial chromosome) transgenic mice displays only a slightly reduced phosphorylation of Ser935, whereas a large effect was observed in similar mice harbouring the R1441G variant [37].

Regulation of cellular LRRK2 phosphorylation

The finding that pathogenic LRRK2 mutations lead to cellular LRRK2 dephosphorylation, resulting in altered cellular distribution [12], suggests that maintaining a balanced LRRK2 phosphorylation equilibrium may be essential for its normal function and this is therefore strictly regulated. Hence elucidating the players involved in this regulation as well as the role for LRRK2 in controlling its own cellular phosphorylation level has become the focus of intense investigation.

Pharmacological inhibition of LRRK2 kinase activity, induced by LRRK2-IN1, H-1152 or sunitinib, leads to cellular LRRK2 dephosphorylation and impaired 14-3-3 binding, resulting in LRRK2 relocalization [9,11]. This suggests a prominent role for LRRK2 kinase activity in its own phosphorylation. However, studies with functional mutants have added new insights. Several studies using kinase-dead mutations show a similar cellular phosphorylation level compared with WT LRRK2, at least at the most established cellular phosphorylation sites [10,12] (Table 1). The same result is observed for overall cellular phosphorylation of kinase-dead LRRK2 measured via metabolic phospholabelling [39], although reduced cellular phosphorylation is seen in truncated kinase-dead constructs [8] or in Thr1410 phosphorylation of the D1994N LRRK2 kinase-dead variant [28]. Moreover, the most common pathogenic mutation G2019S, which clearly shows enhanced kinase activity, does not lead to an enhanced cellular phosphorylation level, calling into question the idea that LRRK2 kinase activity is the major autoregulatory mechanism for cellular LRRK2 phosphorylation. Besides kinase activity, evidence for a prominent role for the GTP domain in cellular LRRK2 phosphorylation is also missing. The functional mutations T1348N and K1347A, resulting in impaired GTP binding, GTPase and kinase activity, display a strongly reduced cellular phosphorylation [40], comparable with the phosphorylation level of pathogenic mutations in the ROC and COR (C-terminal of ROC) domain, which show normal GTP binding, but decreased GTPase activity, compared with WT LRRK2 [12]. However, the pathogenic mutation I2020T, with GTP binding, GTPase and kinase activity levels comparable with WT LRRK2, also leads to impaired cellular phosphorylation, suggesting further that LRRK2 kinase or GTPase activity does not play a prominent role in the regulation of its own cellular phosphorylation. Indeed, a closer look at LRRK2 GTPase and kinase activity on the one hand and cellular LRRK2 phosphorylation on the other reveals that the degree of GTPase or in vitro kinase activity does not correlate with the level of cellular phosphorylation when multiple disease mutants are compared (Table 2). This, together with the observation that autophosphorylation sites clearly differ from the cellular phosphorylation sites, support further the idea that cellular LRRK2 phosphorylation is mediated by other kinases and does not depend on its own enzymatic activity.

On the basis of this notion, it can be hypothesized that several regulatory proteins are involved in regulating LRRK2 cellular phosphorylation probably in the form of regulatory signalling cascades, including at least one kinase and one phosphatase. Although no LRRK2 phosphorylation regulators are currently confirmed, a literature scan for reports of proteins known to be involved in phosphorylation events and which have been linked to LRRK2 via different methods yields a substantial list of potential LRRK2-associated phosphorylation proteins (Table 3). For instance, on the basis of the cellular phosphorylation motif in LRRK2, PKA (protein kinase A) was predicted to act as an upstream kinase [27], and this was explored further by cellular experiments showing reduced LRRK2 phosphorylation after inhibition of PKA [39]. The findings that PKA phosphorylates the cellular LRRK2 phosphorylation site Ser935in vitro together with increased Ser935 phosphorylation after overexpression or stimulation of PKA support further a role for PKA in cellular LRRK2 phosphorylation [37]. Other phosphorylation regulators which have arisen in studies of LRRK2 candidate substrates, candidate-interacting proteins, LRRK2-dependent differential gene expression or phosphorylation motif studies are listed in Table 3. This list is speculative as studies confirming the direct involvement of these proteins in regulating cellular phosphorylation of LRRK2 are lacking. Also, studies screening for regulators of LRRK2 cellular phosphorylation have yet to be reported.

Table 3
Overview of proteins linked to LRRK2 and involved in phosphorylation and dephosphorylation

Proteins reported at least twice are depicted in bold. AK, adenylate kinase; AKAP, A-kinase-anchoring protein; AMPK, AMP-activated protein kinase; ATM, ataxia telangiectasia mutated; CaMKK2, Ca2+/calmodulin-dependent protein kinase kinase 2; CIT, citron (Rho-interacting serine/threonine kinase); CK, casein kinase; DAPK, death-associated protein kinase; ERK, extracellular-signal-regulated kinase; FLJ, hypothetical protein; GS, G2019S; GSK, glycogen synthase kinase; HEK, human embryonic kidney; JIP, JNK (c-Jun N-terminal kinase)-interacting protein; KD, knockdown; MAPK, mitogen-activated protein kinase; MARKK, microtubule affinity-regulating kinase-activating kinase; MET, hepatocyte growth factor tyrosine kinase receptor; MKK, mitogen-activated protein kinase kinase; MTMR, myotubularin-related protein; Nek, never in mitosis gene a-related kinase; PDK, phosphoinositide-dependent protein kinase; PINK, phosphatase and tensin homologue deleted on chromosome 10-induced protein kinase 1; PKB, protein kinase B; PKC, protein kinase C; PPP, phosphoprotein phosphatase; PRKDC, DNA-dependent protein kinase catalytic subunit; PTPN, protein tyrosine phosphatase, non-receptor-type; RIOK, RIO kinase; RIPK, receptor-interacting protein kinase; RPS6KA, ribosomal protein S6 kinase, 90 kDa polypeptide; TAOK, thousand-and-one amino acids kinase; ULK, unco-ordinated 51-like kinase; uMtCK, ubiquitous mitochondrial creatine kinase; WNK, with no lysine; ZAK, sterile α-motif- and leucine zipper-containing kinase.

Protein Identification Potential role Reference(s) 
Akt1 (PKB) In vitro kinase assay and LRRK2 KD effects Substrate [47
Cdc25A Contains consensus phosphorylation motif Substrate [28
LRRK2 In vitro kinase assay Substrate [1515,1919 ] 
MARKK In vitro kinase assay (GS LRRK2) Substrate [48
MKK3/4/6/7 In vitro kinase assay (GS LRRK2) Substrate [49
MKK3/6/7 In vitro kinase assay Substrate [50
RIPK2 Protein array with GS LRRK2 Substrate [48
Serine/threonine protein kinase 3/24/25 Protein array with GS LRRK2 Substrate [48
Serine protein kinase ATM Contains consensus phosphorylation motif Substrate [28
Serine/threonine protein kinase Nek3 Contains consensus phosphorylation motif Substrate [28
Serine/threonine protein kinase ULK1 Contains consensus phosphorylation motif Substrate [28
Serine/threonine protein kinase WNK4 Contains consensus phosphorylation motif Substrate [28
TAOK3 Protein array with GS LRRK2 Substrate [48
CK2 Predicted phosphorylation sequence in LRRK2 Upstream kinase [27
PKA Predicted phosphorylation sequence in LRRK2 Upstream kinase [2727 ] 
 In vitro kinase assay and cellular evidence Upstream kinase [2626,3737 ] 
PKCζ In vitro kinase assay Upstream kinase [48
Serine protein kinase ATM Predicted phosphorylation sequence in LRRK2 Upstream kinase [27
Akt1 Pull-down Interactor [47
AKAP8 Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [51
DAPK1 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [52
JIP1–JIP4 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [53
LRRK1 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [52,54
LRRK2 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [1515,1818,2525,5252,5555,5656 ] 
MKK3/6/7 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [50
PKCζ Protein array with GS LRRK2 and endogenous interaction in mouse brain Interactor [48
PPP2R1A Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [41
PRKDC Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [51
PTPN23 Yeast two-hybrid Interactor [43
Serine protein kinase 1 Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [51
Serine/threonine kinase 24/25 Protein array with GS LRRK2 Interactor [48
Sgg (GSK3β homologue) Co-immunoprecipitation after LRRK2 overexpression in Drosophila melanogaster Interactor [57
uMtCK Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [58
AK2 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
AMPK LRRK2 overexpression enhances AMPK phosphorylation in HEK-293T cells Link with LRRK2 [59
CaMKK2 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
CIT Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
ERK1/2 LRRK2 overexpression enhances ERK phosphorylation in HEK-293T cells Link with LRRK2 [6060 ] 
 LRRK2 overexpression affects ERK phosphorylation dependent on conditions in HEK-293T cells Link with LRRK2 [6161 ] 
FLJ25006 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
FLJ39207 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
MAPK8 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
MET Selected kinase score in renal cell carcinoma Link with LRRK2 [62
MTMR12 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PDK3 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PINK1 Phenotypic interaction in Drosophila melanogaster Link with LRRK2 [6262 ] 
 Phenotypic interaction in Caenorhabditis elegans Link with LRRK2 [6363 ] 
PPP1R12B Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PPP2R2A Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PPP6C Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
RIOK2 (yeast) Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
RPS6KA3 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
ZAK Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
Protein Identification Potential role Reference(s) 
Akt1 (PKB) In vitro kinase assay and LRRK2 KD effects Substrate [47
Cdc25A Contains consensus phosphorylation motif Substrate [28
LRRK2 In vitro kinase assay Substrate [1515,1919 ] 
MARKK In vitro kinase assay (GS LRRK2) Substrate [48
MKK3/4/6/7 In vitro kinase assay (GS LRRK2) Substrate [49
MKK3/6/7 In vitro kinase assay Substrate [50
RIPK2 Protein array with GS LRRK2 Substrate [48
Serine/threonine protein kinase 3/24/25 Protein array with GS LRRK2 Substrate [48
Serine protein kinase ATM Contains consensus phosphorylation motif Substrate [28
Serine/threonine protein kinase Nek3 Contains consensus phosphorylation motif Substrate [28
Serine/threonine protein kinase ULK1 Contains consensus phosphorylation motif Substrate [28
Serine/threonine protein kinase WNK4 Contains consensus phosphorylation motif Substrate [28
TAOK3 Protein array with GS LRRK2 Substrate [48
CK2 Predicted phosphorylation sequence in LRRK2 Upstream kinase [27
PKA Predicted phosphorylation sequence in LRRK2 Upstream kinase [2727 ] 
 In vitro kinase assay and cellular evidence Upstream kinase [2626,3737 ] 
PKCζ In vitro kinase assay Upstream kinase [48
Serine protein kinase ATM Predicted phosphorylation sequence in LRRK2 Upstream kinase [27
Akt1 Pull-down Interactor [47
AKAP8 Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [51
DAPK1 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [52
JIP1–JIP4 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [53
LRRK1 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [52,54
LRRK2 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [1515,1818,2525,5252,5555,5656 ] 
MKK3/6/7 Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [50
PKCζ Protein array with GS LRRK2 and endogenous interaction in mouse brain Interactor [48
PPP2R1A Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [41
PRKDC Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [51
PTPN23 Yeast two-hybrid Interactor [43
Serine protein kinase 1 Co-immunoprecipitation after LRRK2 overexpression in HEK-293T cells Interactor [51
Serine/threonine kinase 24/25 Protein array with GS LRRK2 Interactor [48
Sgg (GSK3β homologue) Co-immunoprecipitation after LRRK2 overexpression in Drosophila melanogaster Interactor [57
uMtCK Co-immunoprecipitation after co-expression in HEK-293T cells Interactor [58
AK2 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
AMPK LRRK2 overexpression enhances AMPK phosphorylation in HEK-293T cells Link with LRRK2 [59
CaMKK2 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
CIT Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
ERK1/2 LRRK2 overexpression enhances ERK phosphorylation in HEK-293T cells Link with LRRK2 [6060 ] 
 LRRK2 overexpression affects ERK phosphorylation dependent on conditions in HEK-293T cells Link with LRRK2 [6161 ] 
FLJ25006 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
FLJ39207 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
MAPK8 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
MET Selected kinase score in renal cell carcinoma Link with LRRK2 [62
MTMR12 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PDK3 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PINK1 Phenotypic interaction in Drosophila melanogaster Link with LRRK2 [6262 ] 
 Phenotypic interaction in Caenorhabditis elegans Link with LRRK2 [6363 ] 
PPP1R12B Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PPP2R2A Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
PPP6C Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
RIOK2 (yeast) Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
RPS6KA3 Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42
ZAK Microarray after LRRK2 KD in SH-SY5Y cells Link with LRRK2 [42

It is noteworthy that the fast effect seen after LRRK2 kinase inhibitor treatment, namely LRRK2 dephosphorylation within 30 min [11], suggests a predominant role for phosphatases in the phosphorylation–dephosphorylation equilibrium of cellular LRRK2 phosphorylation sites. However, to date, no phosphatases have been proven to affect LRRK2 phosphorylation in the cell. Information on the potential role of phosphatases of LRRK2 include the presence of regulatory subunit 1 of protein phosphatase 2A (PPP2R1A) in an LRRK2 immunoprecipitation complex, the identification of protein tyrosine phosphatase 23 as an interactor in a yeast two-hybrid screen and the differential transcript expression of a regulatory subunit of protein phosphatase 1 and 2A (PPP1R12B and PPP2R2A), the catalytic core of protein phosphatase 6 (PPP6C) and MTMR12 (myotubularin-related protein 12) after LRRK2 knockdown; however, none of these candidate proteins has been investigated further [4143].

In summary, accumulating evidence suggests that understanding cellular LRRK2 phosphorylation is crucial to obtain more insight into the function as well as dysfunction of LRRK2. However, to date, many questions remain unanswered. If LRRK2 kinase activity is not the regulator of its own phosphorylation in the cell, then what is the main function of LRRK2 kinase activity and under which cellular conditions, if any, does LRRK2 autophosphorylation occur? If it is not by LRRK2 kinase activity, then how is cellular LRRK2 phosphorylation regulated, and is LRRK2 itself involved in this regulation? Is it possible that conformational changes induced by mutations, rather than effects on LRRK2 activity, make LRRK2 more sensitive to phosphatases or less accessible for kinases? Taking into consideration that cellular consequences of pharmacological LRRK2 kinase inhibition look similar to cellular effects of several pathogenic mutants, is targeting LRRK2 kinase activity still a promising therapeutic strategy?

Conclusions

Although LRRK2 possesses distinct phosphorylation activity in vitro, the lack of confirmed physiological substrates and of prominent LRRK2 autophosphorylation activity in cells raises the question of what the physiological role of LRRK2 kinase activity is. In contrast, extensive evidence now confirms cellular LRRK2 phosphorylation which is moreover disturbed in pathogenic mutants as well as after LRRK2 kinase inhibitor treatment. However, the effectors involved in the regulation of cellular LRRK2 phosphorylation are still unknown. Therefore the identification of regulators of cellular LRRK2 phosphorylation is currently a major challenge in order to improve our understanding of LRRK2 function and dysfunction. These regulators may point to the LRRK2 cellular signalling pathway, how this pathway is altered by disease mutants and how it can be targeted for potential therapy.

LRRK2: Function and Dysfunction: A Biochemical Society Focused Meeting held at Royal Holloway, University of London, Egham, UK, 28–30 March 2012. Organized and Edited by Patrick Lewis (University College London, U.K.) and Dario Alessi (Dundee, U.K.).

Abbreviations

     
  • BAC

    bacterial artificial chromosome

  •  
  • LRR

    leucine-rich repeat

  •  
  • LRRK2

    leucine-rich repeat kinase 2

  •  
  • PD

    Parkinson's disease

  •  
  • PKA

    protein kinase A

  •  
  • ROC

    Ras of complex proteins

  •  
  • WT

    wild-type

Funding

Support from the Michael J. Fox Foundation, the Research Foundation – Flanders (FWO), KULeuven and the Fund Druwé-Eerdekens managed by the King Baudouin Foundation is gratefully acknowledged.

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