Mutations in LRRK2 are among the most common genetic mutations associated with PD. The mechanism by which mutations might contribute to disease is largely unknown, and indeed, the general biology of LRRK2 is poorly understood. In this paper, we set out to understand one aspect of LRRK2 biology evident from its structure. The kinase domain of LRRK2 is homologous to RIP kinases and mixed lineage kinases, which are classes of kinases known to regulate the stress kinase cascades in part through binding to MKKs. We have now shown that LRRK2 also binds to MKKs. We also demonstrate that LRRK2 interacts with a family of scaffold proteins, termed JIPs, which regulate the stress kinase cascades. Cotransfecting LRRK2 increases JIP expression levels, and interaction of LRRK2 with the JIPs might promote their oligomerization, which is thought to be associated with MAPK activation [28
]. Most but not all disease-related mutations in LRRK2 that we studied exhibited high-molecular-weight bands indicative of oligomerization. Interestingly, cotransfecting LRRK2 with JIP4, and possibly other JIPs, also increases levels of ubiquitinated JIP4, although the significance of this change is unclear.
Our observation that LRRK2 associates with MKKs and with JIPs is consistent with an increasing body of evidence suggesting that LRRK2 regulates the stress kinase cascade. Studies in Caenorhabditis elegans
demonstrate that LRRK2 regulates the response to mitochondrial stress (complex I inhibition), and that the response requires the activity of the nematode homologues of MKK6 and p38 [27
]. LRRK2 is known to autophosphorylate, although the amount of autophosphorylation is very low. Gloeckner et al. [26
] recently demonstrated that LRRK2 can transphosphorylate MKK 3–7 in vitro. The reaction does not appear to be catalytic, though, because levels of MKK phosphorylation were similar to levels of LRRK2 autophosphorylation, despite addition of more than a 10,000-fold excess of MKK. Phosphorylation of MKK by LRRK2 occurs in the amino-domain, which is far from the sites normally associated with MKK activation [26
]. The low level of phosphorylation is consistent with a model in which LRRK2 binds the MKKs, phosphorylates the proteins, but then either does not release the MKK or becomes catalytically inactive. Studies from our laboratory demonstrate that LRRK2 binds to MKKs, but interestingly, we find evidence for phosphorylation of MKKs by LRRK2 in vitro, but not in vivo [27
]. Our results suggest that binding of LRRK2 to MKK2 increases the expression and membrane localization of both proteins, as shown by immunocytochemistry and biochemical fractionation.
The interaction of LRRK2 with JIPs is consistent with the observed interaction between LRRK2 and MKKs [27
]. JIPs are scaffold proteins for the stress kinase cascade [28
]. The ability of LRRK2 to bind JIPs in absence of cotransfected MKKs suggests that LRRK2 binds JIPs directly, or at least through a mechanism that does not require MKKs. Thus, the JIP scaffolding complex appears to contain binding sites for both LRRK2 and MKKs. JIPs function to bring together MKKs with MAPKs (e.g. JNK and p38), and also to direct binding to different sites within the cells. Some JIPs tend to direct binding towards the membrane, such as JIP4, while JIP1–3 are known to bind to microtubules. The interaction of LRRK2 with JIP1–3 suggests that LRRK2 might bind to JIP1–3 as a cargo protein, and that binding might facilitate transport along microtubules. Such transport might be particularly important in neurons where LRRK2 can be found in synaptic fields, such as in the striatum. Other proteins linked to disease also appear to interact with JIPs. α-Synuclein was recently shown to regulate levels of JIP1 [34
]. Amyloid precursor protein binds to JIP1 in much the same manner, although the biology of the interaction remains unclear [35
LRRK2 exhibits a striking ability to increase total levels of JIPs and ubiquitination of JIPs. These two effects in combination suggest that LRRK2 inhibits degradation of JIPs, either through the proteasomal or autophagic systems. Alternatively, LRRK2 might also act to increase JIP translation or transcription. A link between LRRK2 and regulation of protein translation is suggested by the observation that LRRK2 binds and phosphorylates 4E-BP, a regulator of protein translation machinery [24
]. A putative role in regulating protein degradation is consistent with the presence of ubiquitin-positive inclusions in most cases of PD that are associated with LRRK2 mutations [5
]. Most of these inclusions contain α-synuclein, but some inclusions contain tau protein [5
]. The putative ability of LRRK2 to inhibit autophagy or proteasomal function could contribute to increased risk of PD and inclusion formation among subjects with LRRK2 mutations. Several reports also note that LRRK2 overexpression leads to the formation of inclusions containing LRRK2 [20
]. Other reports indicate that LRRK2 regulates autophagy [38
]. Inhibition of autophagy or proteasomal function could induce inclusion, stimulate inclusion formation as well as increase levels of JIP and JIP oligomers. Further experiments are needed to determine the precise mechanism.
In summary, our data suggest that a strong biological connection exists between LRRK2, the stress kinase cascade and JIPs, a class of scaffold proteins that bind stress kinases. The role of stress kinases in cell death creates a cogent linkage between LRRK2 and cell death. Whether this pathway is the actual pathway linked to the pathophysiology of disease among subjects carrying mutations in LRRK2 remains to be determined. Most of the mutant LRRK2 constructs increased JIP4 levels and oligomerization more than WT with the exception of Y1699C. The incomplete correlation between pathological LRRK2 mutations and enhanced JIP expression/oligomerization suggests that this axis is not the principle mechanism by which LRRK2 causes disease. However, understanding the mechanisms by which LRRK2 modulates the stress kinase cascade could lead to increased understanding of the biology of LRRK2 and potential differences in the pathophysiology of disease among individuals with different LRRK2 mutations.