Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site (¹⁴⁴¹RASpS¹⁴⁴⁴). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.Parkinson disease (PD), one of the most prevalent neurodegenerative afflictions, is characterized pathologically by the selective loss of dopaminergic neurons in the midbrain and by the presence of intracellular inclusions in the remaining cells, termed Lewy bodies (1). However, the molecular mechanisms underlying the complex pathological process are poorly understood. Many genetic and environmental factors contribute to the disease, and mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD. LRRK2 is a large protein of 285 kDa and encodes several structural motifs, such as armadillo, ankyrin, and the namesake leucine-rich repeats, a Ras of complex proteins (ROC) GTPase, a C-terminal of ROC (COR), a kinase domain [with sequence homology to MAP kinase kinase kinase (MAPKKKs)], and a C-terminal WD40 domain (2). Notably, mutations known to cause PD are located within the catalytically active GTPase (ROC) and kinase domains of LRRK2 (see Fig. 3A) (3). Particularly for a single residue located within the ROC domain, three independent PD-associated mutations (R1441C, R1441G, and R1441H) have been found (4), whereas the kinase domain may harbor the most frequent pathogenic mutation, G2019S. Mutations at both these sites have been associated with enhanced kinase activity compared with wild type (5, 6), suggesting that dysregulation of these enzymatic activities may contribute to PD pathogenesis.Open in a separate windowFig. 3.S1444 on LRRK2 is a PKA-induced 14-3-3 binding site. (A) Multidomain structure of LRRK2. ANK, ankyrin repeat region; LRR, leucine-rich repeat domain; ROC, Ras of complex (GTPase); COR, C-terminal of ROC. The potential 14-3-3 interaction motif in LRRK2 is shown and aligned to a mode I 14-3-3 binding consensus sequence. Known pathogenic mutations R1441C/G/H and G2019S are indicated. (B) LRRK2 pull-down with recombinant GST–14-3-3 gamma. LRRK2 WT and LRRK2 WT ∆967 were expressed in Sf9 cells. Lysates were incubated with GST–14-3-3 gamma for 4 h. GST–14-3-3 gamma pulled down both LRRK2 WT full length and LRRK2 WT ∆967. Samples were separated on a 4–12% gradient SDS gel, and the membranes were probed with Strep-Tactin HRP or GST antibody. (C) LRRK2 WT ∆967 was precipitated with GST–14-3-3-agarose in the absence or in the presence of increasing concentrations of chemically synthesized LRRK2 peptides (LFNIKARASSSPVILVGT) phosphorylated or nonphosphorylated at S1444. Sample separation and Western blotting were carried out as described under B. (D) Two micrograms of His-ROC WT, S1443A, S1444A, or S1443A-S1444A mutant proteins was incubated in the presence or absence of PKA for 1 h at 30 °C. Far western blotting was performed using GST–14-3-3 protein as a probe. PKA-induced ROC–GST–14-3-3 interaction could be observed only when S1444 was present. Equal loading was demonstrated using anti-His antibody.LRRK2 is a cytosolic phosphoprotein (7) phosphorylated in vitro by a variety of serine/threonine kinases, including PKC zeta (8), serine protein kinase ataxia telangiectasia mutated (9), the IκB kinase family (10), and cAMP-dependent protein kinase (PKA) (11, 12). PKA is a key regulator of a vast number of signaling molecules and is critical for neuronal functions such as synaptic plasticity, protein trafficking, protein degradation, neuronal excitability, and regulation of dopamine physiology (13–17). In LRKK2, the conserved residue (S935) was shown to be phosphorylated by PKA (12), and recently this site was proposed as a biomarker for LRRK2 activity (10, 18, 19). Phosphorylation of S910 and S935 within LRRK2 promotes binding of 14-3-3 proteins (19), a family of small 29–30 kDa acidic regulatory proteins, highly conserved and ubiquitously expressed in various tissues. The binding of 14-3-3 proteins results in various downstream effects, such as changes in structural conformations, kinase activity, and subcellular localization of the target proteins (20, 21). Nichols et al. (19) proposed a role for 14-3-3 proteins in regulating the cytoplasmic localization of LRRK2, whereas Li et al. (12) observed protection from dephosphorylation of S935 after 14-3-3 binding. Furthermore, recent data from Fraser et al. (22) suggest a regulatory function of 14-3-3 binding in controlling extracellular release of LRRK2. Dysregulation of 14-3-3/client protein interaction has been shown to facilitate the development of several human disorders (23, 24), and an influence of the pathogenic mutation R1441G on LRRK2/14-3-3 interaction has been demonstrated (12, 19). Although these data support an involvement of PKA and 14-3-3 proteins in regulating LRRK2 function, LRRK2 phosphorylation by PKA, as well as 14-3-3 binding and possible (patho)physiological consequences of this interplay, have not yet been addressed in detail.Here, we systematically mapped PKA phosphorylation sites in LRRK2 and further investigated the impact of this phosphorylation in terms of 14-3-3 binding and LRRK2 function. Our results predict an essential function for PKA phosphorylation and subsequent 14-3-3 interaction in the negative regulation of LRRK2 kinase activity.