Here we report the identification of CHIP as a LRRK2 interacting protein and provide evidence that CHIP can significantly reduce the cellular levels of LRRK2 by ubiquitination and proteasome-dependent degradation. Our data reveal which domains of CHIP and LRRK2 are required or dispensable for this interaction. Surprisingly, we found that CHIP can bind to LRRK2 at least two independent ways. The combined data shown in and indicate that the charged domain of CHIP binds to the ROC domain of LRRK2 and that the TPR domain of CHIP is required for binding to the N-terminal region of LRRK2 that includes the armadillo, ankyrin and leucine-rich repeats. This is the first demonstration that the charged domain of CHIP directly binds to another protein. The TPR domain of CHIP apparently binds to the N-terminal region of LRRK2 indirectly via Hsp90, which is known to bind to the TPR domain of CHIP 
, because blocking Hsp90 function with geldanamycin completely prevented the binding of full-length CHIP to the N-terminal region of LRRK2. By contrast, geldanamycin did not prevent the binding of full-length CHIP to the C-terminal half of LRRK2 (), indicating that the interaction between the charged domain of CHIP and the ROC domain of LRRK2 does not require Hsp90 function. We further show that the isolated TPR domain of CHIP is sufficient for binding to the N-terminal half of LRRK2, but not to the C-terminal half of LRRK2 (Supplemental Figure S2
). Consistent with the geldanamycin data, the K30A point mutation within the TPR domain that blocks CHIP binding to Hsp90, also blocks binding to the N-terminal half of LRRK2 (Supplemental Figure S2
We propose a model in which the charged domain of CHIP binds to the ROC domain of LRRK2 either directly or indirectly, but independent of Hsp90, while the TPR domain of CHIP binds to the N-terminus of LRRK2 indirectly, via Hsp90 (). Surprisingly, the U-box domain of CHIP, which is structurally related to RING finger ubiquitin ligase domains, is apparently dispensable for CHIP binding to LRRK2 (). This mode of binding differs from the previous finding that LRRK2 binds to the second RING finger domain of the E3 ubiquitin ligase Parkin 
. LRRK2 reportedly enhances the auto-ubiquitination activity of Parkin, however, Parkin does not ubiquitinate LRRK2 
. By contrast, our data indicates that CHIP ubiquitinates LRRK2 (). Because CHIP has also been reported to form a complex with Parkin and to enhance the ubiquitin ligase activity of Parkin 
, it is possible that the reported interaction between LRRK2 and Parkin is mediated by CHIP.
Our most striking finding is that CHIP decreased the stability of LRRK2 in a dose-dependent manner (). Because we found that CHIP can bind directly to LRRK2 and cause the ubiquitination and proteasome-dependent degradation of LRRK2, this is a likely mechanism by which CHIP destabilizes LRRK2. CHIP-mediated degradation of LRRK2 appears to require the chaperone interaction function of CHIP because we did not observe any LRRK2 destabilization by a TPR domain point mutant of CHIP (K30A) or by a TPR domain deletion mutant of CHIP (CHIPΔT), both of which are unable to interact with Hsp/Hsc70 or Hsp90 
. CHIP-mediated degradation of LRRK2 appears to also require the ubiquitin ligase activity of CHIP because we did not observe any LRRK2 destabilization by a U-box domain point mutant of CHIP (H260Q) or by a U-box domain deletion mutant of CHIP (CHIPΔU), both of which are unable to catalyze protein ubiquitin conjugation 
Our studies showed similar CHIP-mediated degradation of wild-type LRRK2 and LRRK2 with disease-linked point mutations (), therefore the pathogenic mechanisms of the mutations we tested do not likely involve diminished CHIP-mediated degradation. These findings are consistent with previous studies that found no effect of LRRK2 mutations on ubiquitination, proteasomal degradation, steady-state levels or turnover of LRRK2 
. However, we found that the kinase activity of LRRK2 is important for stabilizing cellular levels of LRRK2 because an artificial variant of LRRK2 lacking kinase activity was markedly and consistently destabilized even in the absence of exogenous CHIP (). The diminished abundance of R1441C and D1994A LRRK2 in the absence of exogenous CHIP could be due to degradation mediated by endogenous CHIP, other degradation mechanisms or to other possible effects such as altered expression. LRRK2 kinase activity does not appear to be required for CHIP mediated-degradation because CHIP decreased the abundance of the artificial kinase-inactive mutant (D1994A) LRRK2 in a dose-dependent manner (), although this experiment cannot rule out the possibility that CHIP affects expression rather than degradation.
CHIP has previously been shown to promote the proteasomal degradation of other proteins implicated in neurodegenerative diseases such as tau, Aβ, α-synuclein oligomers and proteins with expanded polyglutamine repeats 
. During revision of this manuscript, Ko et al.
reported that CHIP regulates LRRK2 ubiquitination, degradation and toxicity 
. Similar to our findings, Ko et al.
found that CHIP binds to both wild-type and mutant LRRK2 and promotes the ubiquitination and proteasomal degradation of LRRK2. Our findings differ in that we identified two independent means of CHIP binding to LRRK2: an indirect interaction between the TPR domain of CHIP and the ARM domain of LRRK2, likely via Hsp90, and an interaction between the charged domain of CHIP and the ROC domain of LRRK2, which is either direct or indirect via a common adaptor protein. The ARM domain interaction may not have been detected by Ko et al.
because the isolated full length ARM domain was not shown to be tested for CHIP binding.
In addition to CHIP, our yeast two-hybrid analysis also identified Hsp90 as a LRRK2 binding protein. Several previous studies have shown that Hsp90 can bind to full length LRRK2 or to the kinase domain of LRRK2 
. Here we show for the first time that Hsp90 can also bind to the ARM domain at the N-terminus of LRRK2. ARM domains mediate protein-protein interactions and are found in proteins with diverse functions including cytoskeletal regulation and intracellular signaling 
. Here we propose a model in which the ARM domain stabilizes LRRK2 in part by binding to Hsp90 (). Because the TPR domain of CHIP is well-known to bind to Hsp90 
, it is likely that a portion of the LRRK2-Hsp90 complex will also be associated with CHIP. LRRK2 apparently forms a stable complex with Hsp90 and CHIP because we successfully immunoprecipitated endogenous CHIP and endogenous Hsp90 together with LRRK2 ().
Our data () confirm that overexpression of Hsp90 stabilizes LRRK2 and that inhibition of endogenous Hsp90 by geldanamycin destabilizes LRRK2, as previously reported 
. Importantly, we show for the first time that Hsp90 overexpression impairs CHIP-mediated degradation of LRRK2 () and that inhibition of endogenous Hsp90 by geldanamycin enhances CHIP-mediated degradation of LRRK2 (). According to our model (), inhibition of endogenous Hsp90 by geldanamycin prevents CHIP binding to the N-terminal half of LRRK2 () but does not inhibit the Hsp90-independent binding of CHIP to the C-terminal half of LRRK2 (including the ROC domain), which promotes the ubiquitination and proteasome-dependent degradation of LRRK2. In the presence of excess CHIP, Hsp90-dependent binding of CHIP to the ARM domain of LRRK2 can also destabilize LRRK2. We hypothesize that the stability of LRRK2 depends on the ratio of the cellular abundance and binding availability of Hsp90 and CHIP.
The fact that LRRK2 mutations cause both familial and apparently sporadic forms of PD with typical clinical symptoms and late age-at-onset 
highlights the significance of age as a causative factor for both familial and idiopathic PD. An important question is whether the cellular abundance and binding availability of Hsp90 and CHIP change with age and whether this contributes to PD risk, perhaps by increasing LRRK2 abundance, aggregation or neurotoxicity. It has been proposed that the ability of CHIP to degrade potentially neurotoxic misfolded, damaged or mutated proteins might diminish with age 
. Our findings raise the possibility that diminished CHIP-mediated degradation of LRRK2 in aged or stressed neurons may contribute to sporadic PD as well as familial PD in patients bearing LRRK2 mutations. Further studies of the detailed mechanisms that control LRRK2 levels may lead to the development of PD therapies that exploit these mechanisms to degrade wild-type or mutant LRRK2 and thereby mitigate neurotoxicity.