Regulation of LRRK2 stability by the E3 ubiquitin ligase CHIP

Abstract

Dominantly inherited mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common cause of familial Parkinson's disease (PD) and have also been identified in individuals with sporadic PD. Although the exact cellular function of LRRK2 remains unknown, most PD-linked mutations appear to be toxic to cells in culture via mechanisms that depend on the kinase activity of LRRK2 or on the formation of cytoplasmic inclusions. Here we show that the E3 ubiquitin ligase CHIP physically associates with LRRK2 and regulates the cellular abundance of LRRK2. We further show that LRRK2 forms a complex with overexpressed and endogenous CHIP and Hsp90. Our data indicates that the destabilization of LRRK2 by CHIP is due to ubiquitination and proteasome-dependent degradation. Hsp90 can attenuate CHIP-mediated degradation and this can be blocked by the Hsp90 inhibitor geldanamycin. These findings provide important insight into the cellular regulation of LRRK2 stability and may lead to the development of therapeutics to treat PD based on controlling LRRK2 stability.

Figure 1. Co-immunoprecipitation of partial and full-length LRRK2, CHIP and Hsp90.

The indicated myc- and HA-epitope tagged constructs (0.5 µg for each) were co-transfected into 4.5×10⁶ HEK293 cells in 60 mm dishes. Lactacystin was added to the medium at a final concentration of 5 µM to prevent CHIP-mediated degradation of LRRK2. HEK293 cell lysates were subjected to immunoprecipitation using polyclonal anti-myc or anti-HA antibodies, then immunoblotted using monoclonal anti-HA or anti-myc antibodies, respectively. Cell lysates were also directly immunoblotted using monoclonal anti-HA and anti-myc antibodies to verify similar protein expression levels in experimental and empty expression vector (CMV) control transfections. (A) The ROC domain of LRRK2 co-immunoprecipitates with its interactor identified from the yeast two-hybrid screen, CHIP_150–200. (B) The ARM domain of LRRK2 co-immunoprecipitates with its interactor identified from the yeast two-hybrid screen, Hsp90_248–stop. (C) Full-length LRRK2 co-immunoprecipitates with full-length CHIP. (D) Full-length LRRK2 co-immunoprecipitates with full-length Hsp90.I

Figure 2. CHIP binds to multiple domains of LRRK2 by different mechanisms.

The indicated constructs (0.5 µg for each) were co-transfected into 4.5×10⁶ HEK293 cells in 60 mm dishes. Lactacystin was added to the medium at a final concentration of 5 µM to prevent CHIP-mediated degradation of LRRK2. HEK293 cell lysates were subjected to immunoprecipitation using polyclonal anti-myc or anti-HA antibodies, then immunoblotted using monoclonal anti-HA or anti-myc antibodies, respectively. Cell lysates were also directly immunoblotted using monoclonal anti-HA and anti-myc antibodies to verify similar protein expression levels in experimental and empty expression vector (CMV) control transfections. (A) Co-immunoprecipitation of full-length CHIP and the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (B) Co-immunoprecipitation of full-length CHIP and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2; (C) Co-immunoprecipitation of CHIP lacking the U-box domain (CHIPΔU) and the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (D) Co-immunoprecipitation of CHIP lacking the U-box domain (CHIPΔU) and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2; (E) Absence of co-immunoprecipitation of CHIP lacking the TPR domain (CHIPΔT) and the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (F) Co-immunoprecipitation of CHIP lacking the TPR domain (CHIPΔT) and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2; (G) Geldanamycin impairs the interaction of full-length CHIP with the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (H) Geldanamycin does not disrupt the interaction of full-length CHIP and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2. For (G-H), geldanamycin was added into the medium to 1 µM final concentration and incubated for 1 hour prior to cell collection.

Figure 3. CHIP promotes LRRK2 degradation.

(A) CHIP promotes full-length LRRK2 degradation. 1.0 µg of pMyc-LRRK2 was co-transfected with 0, 0.1 0.4, 0.6 or 1.0 µg of pHA-CHIP into 1.5×10⁶ HEK293 cells in 35 mm dishes. Empty vector pHA-CMV was used to normalize the total amount of DNA for each transfection. After 48-hour incubation, equal amounts of cell lysates were used for immunoblotting using anti-myc antibody and the membrane was re-probed using anti-Hsp90, anti-CHIP and anti-β-actin antibodies. (B) Effects of CHIP on the stability of N- and C-terminal portions of LRRK2. 1.0 µg of pMyc-AAL (ARM-Ankyrin-LRR, the N-terminal portion of LRRK2) or pMyc-RCKW (ROC-COR-Kinase-WD40, the C-terminal portion of LRRK2) were co-transfected with 0, 0.5 or 1.0 µg of pHA-CHIP into 1.5×10⁶ HEK293 cells in 35 mm dishes. Empty vector pHA-CMV was used to normalize the total amount of DNA for each transfection. After 48-hour incubation, equal of amounts of cell lysates were used for immunoblotting using anti-myc antibody (the upper panel for each construct). Membranes were stripped and re-probed using anti-β-actin antibody to confirm equal loading. To rule out non-specific down-regulation of the AAL or RCKW portions of LRRK2, isolated leucine rich repeat domain (pMyc-LRR) and kinase domain (pMyc-kinase) of LRRK2 were similarly co-transfected with increasing amounts of CHIP. These domains were not significantly down-regulated by CHIP. NS, non-specific immunoreactive band above the Myc-tagged kinase domain. (C) CHIP destabilizes wild-type and mutant LRRK2. 1 µg of pMyc-LRRK2, LRRK2(G2019S), LRRK2(R1441C) or LRRK2(D1994A) was co-transfected with 0, 0.1, 0.5 or 1.0 µg of pHA-CHIP into 1.5×10⁶ HEK293 cells in 35 mm dishes. Empty vector pHA-CMV was used to normalize the amount of total DNA for each transfection. After 48-hour incubation, the cells were harvested and lysates were immunoblotted with anti-myc and anti-β-actin antibodies to measure the abundance of wild-type and mutant LRRK2.

Figure 4. Both TPR and U-box domain are required for LRRK2 degradation.

1 µg of pMyc-LRRK2 and 0.5 µg of pHA-CMV (Lane 1 from left to right) or pHA-CHIP (Lane 2) or pHA-CHIP(K30A) (Lane 3) or pHA-CHIP(H260Q) (Lane 4) or pHA-CHIPΔU (Lane 5) or pHA-CHIPΔT (Lane 6) were co-transfected into 1.5×10⁶ HEK293 cells in 35 mm dishes. 48 hours after transfection, cells were harvested and the lysates were immunoblotted with anti-myc (upper panel), anti-β-actin (middle panel) and anti-HA (lower panel).

Figure 5. CHIP can ubiquitinate LRRK2 and promote LRRK2 degradation through the ubiquitin proteasome pathway.

(A) From Lane 1 to Lane 6 (from left to right), the indicated constructs were co-transfected into 1.5×10⁶ HEK293 cells in 35 mm dishes. In Lane 3, lactacystin was added into the medium to a final concentration of 5 µM immediately following cell transfection. After 24-hour incubation, the cell lysates were immunoprecipitated using a polyclonal anti-myc antibody. The immunoprecipitants were immunoblotted with monoclonal anti-UB antibody (upper panel). The corresponding cell lysates were probed using anti-myc and anti-β-actin (middle and lower panels). (B) 1.0 µg of pMyc-LRRK2 was co-transfected with 0.5 µg pHA-CMV or 0.5 µg of pHA-CHIP into 1.5×10⁶ HEK293 cells in 35 mm dishes. After 24 hours, lactacystin was added to the indicated concentration. DMSO was added in Lane 1 as a negative control. The cells were cultured for another 24 hours. The cell lysates were probed using anti-myc and anti-β-actin. (C) 1.0 pMyc-LRRK2 and 0.5 g of pHA-CHIP were co-transfected into HEK293 cells. After 24 hours, lactacystin was added to the final concentration at 5 µM. The cells were sampled at 0, 6, 12, 24 and 36 hours after addition of lactacystin. The cell lysates were probed using anti-myc and anti-β-actin. (D) HEK293 cells were transfected with 1.0 µg pMyc-LRRK2, with or without 250 ng pHA-CHIP, pMT-HA-Ub, or pCS2-UbK0, as indicated. 48 hours after transfection, LRRK2 protein levels were determined by immunoblotting with anti-myc antibody.

Figure 6. Hsp90 can attenuate CHIP-mediated LRRK2 degradation.

(A) 1.0 µg pMyc-LRRK2 and 0.5 µg pHA-CHIP were co-transfected with 0, 0.1, 0.4, 0.6, 1.0 µg pFlag-Hsp90. After 48-hour incubation, the cell lysates were immunoblotted with anti-myc and anti-β-actin antibodies. (B) 1.0 µg of pMyc-LRRK2 was co-transfected with 0.5 pHA-CMV (Lane 1 and 2 from left to right) or 0.5 µg pHA-CHIP (Lane 3–8). 24 hours after cell transfection, GA and lactacystin were added to the indicated concentrations and the cells were cultured for another 24 hours prior to cell collection and western analysis.

Figure 7. LRRK2, CHIP and Hsp90 form a protein complex.

(A). 1 µg of pMyc-LRRK2, pHA-CHIP and pFlag-Hsp90 were co-transfected into 1.5×10⁷ HEK293 cells in 100 mm dishes. Lacatcystin was added at 5 µM to prevent CHIP-mediated degradation of LRRK2. The cells were incubated for 48 hours and the cell lysate was subjected to immunoprecipitatiom using a polyclonal anti-myc antibody or rabbit IgG. The immunoprecipitants and their cell lysates were probed with monoclonal anti-HA, anti-Flag and anti-myc antibodies. (B). 3 µg of pMyc-LRRK2 was transfected into 1.5×10⁷ HEK293 cells in 100 mm dishes. The cells were incubated for 48 hours and the cell lysate was subject to immunoprecipitation using polyclonal anti-myc antibody or rabbit IgG. The immunoprecipitants were probed using anti-CHIP or anti-Hsp90 antibodies. (C) Model of LRRK2 binding to CHIP and Hsp90. CHIP can form a stable complex with LRRK2 by at least two independent protein-protein interactions. The N-terminal TPR domain of CHIP can bind to the N-terminal ARM domain of LRRK2 indirectly via Hsp90. The intermediate charged domain of CHIP can bind to the ROC domain of LRRK2 independent of Hsp90. The U-box domain of CHIP is dispensable for binding to LRRK2 but required for CHIP-mediated ubiquitination and proteasome-dependent degradation of LRRK2.