Studies of LRRK tagged with green fluorescent

Studies of LRRK2 tagged with green fluorescent protein revealed the presence of monomeric LRRK2 in the biotin 100 mg of living cells, while LRRK2 oligomers were found in the proximity of the plasma membrane, suggesting that both oligomerization status and compartmentalization of LRRK2 defines its function [67]. Indeed, the need of dimerization and relocation for LRRK2 activity was demonstrated in different cellular models by several groups [68,69]. So far, the best known interaction partner of LRRK2 is the 14-3-3 adaptor protein. Its interaction not only regulates LRRK2 dimerization status but also its phosphorylation pattern. It is known that the dephosphorylation of constitutive phosphorylation sites such as S910, S935, S955 and S973, located between the Ank domain and the LRR motif, disrupts the interaction between LRRK2 and the 14-3-3 protein, resulting in the formation of LRRK2 inclusions in the cytoplasm [68,70,71]. This pattern can be achieved by serine-to-alanine substitutions, LRRK2 kinase inhibition with chemical compounds or arsenite treatment and oxidative stress induction, but can also be mimicked by most PD-associated LRRK2 mutations [68,70,71]. Interaction with a 14-3-3 adaptor protein seems to play a key role in the regulation of LRRK2 kinase activity. Protein kinase A-mediated phosphorylation of S1444 located in the ROC domain enabled the binding of LRRK2 to 14-3-3 and was associated with reduced LRRK2 kinase activity. Conversely, S1444A or the decreased S1444 phosphorylation observed in R1441C/G mutated LRRK2 led to its dissociation from 14-3-3 complex and elevated kinase activity [72]. Similarly, difopein, the competitive 14-3-3 antagonist peptide, increased the kinase activity of G2019S LRRK2 by abrogating its interaction with 14-3-3 [73]. In contrast, loss of 14-3-3 binding during arsenite stress resulted in decreased LRRK2 kinase activity and GTP binding in vitro [68]. In summary, with respect to LRRK2-14-3-3 protein interaction, the majority of studies support a model in which 14-3-3-bound LRRK2 exist in the cytoplasm in a monomeric, phosphorylated state corresponding to the inactive kinase form. Dephosphorylation, dissociation from the 14-3-3 protein, dimerization and relocation of LRRK2 to membrane compartments all correspond with the active form of LRRK2, which can also be obtained by PD-associated mutations. Based on structural studies carried out on the G2019S ortholog from Dictyostelium discoideum, a different mechanism for LRRK2 activation is however also possible. An additional hydrogen bond between the mutated residue and the regulatory region, which in human LRRK2 corresponds to Q1919 within the kinase domain, appear to stabilize the active conformation of the LRRK2 mutant and to mechanistically contribute to its increased kinase activity [74]. It seems that not only catalytic domains, but also other motifs and the phosphorylation status of LRRK2 are important for its cellular functions [75]. The WD40 domain was shown to mediate interactions of LRRK2 with the microtubules [76]. Recently, Kalogeropulou et al. [77] identified p62 as a novel endogenous interacting partner and downstream target for LRRK2 kinase, whereby the interaction was mediated by the Arm and Ank domains at the N-terminus of LRRK2 and the ZZ domain of p62. Of note, the C-terminal ubiquitin-binding domain of p62 influences the phosphorylation of the ZZ domain by LRRK2. Moreover, p62 plays a role in selective autophagy by binding the ubiquitin conjugates and dragging them for autophagic degradation [25]. The N-terminus of LRRK2 is also crucial for the phosphorylation of downstream targets like p62, Rab7L1, Rab8A and Rab10 [77,78]. Furthermore, p62 phosphorylation by LRRK2 is abolished when its S910/S935 residues are dephosphorylated [77]. In the case of LRRK1, its N-terminal part containing the Ank and LRR motifs was crucial for mediating LRRK1 interaction with the late endosomal/lysosomal protein VAMP7 and directing LRRK1 to the lysosomes during tunicamycin-induced autophagy [21].