Parkin mediates the degradation-independent ubiquitination of Hsp70 - PubMed (original) (raw)

Comparative Study

Parkin mediates the degradation-independent ubiquitination of Hsp70

Darren J Moore et al. J Neurochem. 2008 Jun.

Abstract

Mutations in the parkin gene cause autosomal recessive, juvenile-onset parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. Disease-associated mutations cause a loss-of-function of parkin which may compromise the poly-ubiquitination and proteasomal degradation of specific protein substrates, potentially leading to their deleterious accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro and in cultured cells. Parkin interacts with Hsp70 via its second RING finger domain and mutations in/near this domain compromise Hsp70 ubiquitination. Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor does it promote its proteasomal degradation. Consistent with this observation, Hsp70 levels remain unaltered in brains from parkin-deficient autosomal recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70 levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's disease/dementia with Lewy bodies brains. Parkin mediates the multiple mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent role for this ubiquitin modification. Our observations support a novel functional relationship between parkin and Hsc/Hsp70 and support the notion that parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins either via attachment of alternatively linked poly-ubiquitin chains or through multiple mono-ubiquitination to achieve alternate biological outcomes.

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Figures

Fig. 1. Microtubule-associated protein tau is not a parkin substrate

(a) Co-immunoprecipitation of parkin and four-repeat tau. SH-SY5Y cells expressing FLAG-tagged parkin and myc-tagged tau (WT or FTDP-17-linked P301L mutant) were subjected to IP with anti-myc antibody and IPs and inputs were probed with anti-FLAG or anti-myc antibodies, (b) Parkin fails to ubiquitinate tau. SH-SY5Y cells expressing myc-tagged tau (WT or P301L mutant) and HA-tagged ubiquitin with or without FLAG-tagged parkin were subjected to IP with anti-myc antibody and IPs and inputs were probed with anti-HA or anti-tau antibodies. Notice the distinct lack of tau-ubiquitin conjugates revealed by probing with anti-tau antibody. Separate SDS-PAGE gels containing identical anti-myc IPs were stained with coomassie colloidal blue (lower panel). Arrows indicate the presence of tau or associated endogenous Hsp70 that was subsequently identified by MALDI-TOF mass spectrometry. *denotes IgG heavy chain. Molecular mass markers are indicated in kilodaltons (kDa). Blots are representative of at least two independent experiments.

Fig. 2. Hsp70 is ubiquitinated by parkin

(a) In vivo ubiquitination assay. SH-SY5Y cells expressing combinations of HA-tagged ubiquitin, FLAG-tagged parkin and myc-tagged tau (WT or P301L mutant) were subjected to IP with anti-Hsp70 antibody to isolate endogenous Hsp70 and IPs and inputs were probed with anti-HA and anti-Hsp70 antibodies. In the presence of ubiquitin, parkin ubiquitinates endogenous Hsp70 with up to two ubiquitin (Ub) moieties. Tau is not required for parkin-mediated Hsp70 ubiquitination. (b) CHIP fails to ubiquitinate Hsp70. HEK293 cells co-expressing V5-tagged Hsp70 and HA-tagged ubiquitin together with either FLAG-tagged parkin, myc-tagged CHIP or both proteins were harvested and equivalent detergent-soluble protein fractions were probed with anti-V5 antibody to monitor Hsp70-ubiquitin conjugate formation, or with anti-FLAG and anti-myc antibodies. CHIP alone fails to appreciably ubiquitinate Hsp70 and does not influence parkin-mediated Hsp70 ubiquitination. (c) In vitro ubiquitination assay. Recombinant rabbit E1, human UbcH7 (E2), baculovirus (Bv)-derived human parkin, human Hsp70 and human ubiquitin proteins were incubated together in assay buffer containing ATP for 2 h at 37°C. Reactions were probed with anti-Hsp70, anti-parkin and anti-ubiquitin antibodies. In the presence of all components, Hsp70 is directly modified by parkin with up to four ubiquitin moieties reflected by a corresponding shift in the molecular mass of Hsp70. Parkin auto-ubiquitination is revealed by probing with anti-ubiquitin in the absence of Hsp70. (d) In vitro ubiquitination assay with FLAG-tagged parkin derived by IP from transfected SH-SY5Y cells. Reactions were probed with anti-Hsp70, anti-FLAG and anti-ubiquitin antibodies. Parkin catalyzes the ubiquitination of hsp70 and removal of UbcH7 or parkin impairs Hsp70 ubiquitination. Unmodified Hsp70 is indicated by arrows and ubiquitinated forms of Hsp70 [Hsp70-(Ubn)] or ubiquitin-protein conjugates [(Ub)n] are also indicated. Molecular mass markers are indicated in kilodaltons (kDa). Blots are representative of at least two independent experiments.

Fig. 3. Disease-associated RING2 domain mutations impair parkin-mediated Hsp70 ubiquitination

(a) Hsp70 specifically interacts with the RING2 domain of parkin. SH-SY5Y cells expressing V5-tagged Hsp70 and HA-tagged parkin deletion mutants were subjected to IP with anti-V5 antibody and probed with anti-HA and anti-V5 antibodies, whereas inputs were probed with anti-HA antibody. Hsp70 interacts with full-length parkin and deletion mutants (ΔUbl, IBR-R2 and R2) minimally harboring the RING2 (R2) domain. Image highlighting the domain structure and amino acid position of parkin deletion mutants, the location of disease-associated mutations, and the capacity to interact with Hsp70 (+ or −). (b) In vivo ubiquitination assay with disease-associated parkin variants. SH-SY5Y cells expressing V5-tagged Hsp70, HA-tagged ubiquitin and myc-tagged parkin harboring various disease-associated mutations, were subjected to IP with anti-V5 antibody and probed with anti-HA or anti-V5 antibodies to reveal Hsp70-ubiquitin conjugates, and inputs were probed with anti-HA and anti-myc antibodies. Mutations in the RING2 domain (C431F and P437L), the RING1 domain (T240R) or truncating mutations (Q311X and W453X) markedly impair parkin-mediated Hsp70 ubiquitination. ‘X’ represents the introduction of a premature stop codon for some mutations leading to a truncated parkin species. (c) Interaction of Hsp70 with disease-associated parkin variants. HEK293 cells co-expressing myc-tagged parkin variants (WT, T240R, C431F or P437L) with or without V5-tagged Hsp70, were subjected to IP with anti-V5 antibody and probed with anti-myc or anti-V5 antibodies to reveal interactions, and inputs were probed with anti-myc antibody to control for equivalent loading of parkin variants. The T240R and C431F mutations moderately impair the normal interaction of parkin with Hsp70. Ubiquitinated forms of Hsp70 [Hsp70-(Ub)n] and ubiquitin-protein conjugates [(Ub)n] are indicated. Molecular mass markers are indicated in kilodaltons (kDa). Blots are representative of at least two independent experiments.

Fig. 4. Ubiquitination of Hsp70 by parkin is degradation-independent

(a) Ubiquitinated Hsp70 is not degraded by the proteasome. SH-SY5Y cells expressing HA-tagged ubiquitin with or without FLAG-tagged parkin were treated with or without the proteasomal inhibitor MG132 (5 µM) for 24 h, subjected to IP with anti-Hsp70 antibody, and IPs and inputs were probed with anti-HA and anti-Hsp70 antibodies to monitor Hsp70-ubiquitin conjugate formation. Proteasomal inhibition fails to affect the levels of ubiquitinated Hsp70. (b) Parkin-mediated ubiquitination does not influence the steady-state levels of Hsp70. SH-SY5Y cells expressing HA-tagged ubiquitin and increasing quantities of FLAG-tagged parkin (0.05–1 µg plasmid DNA) were harvested at 48 h post-transfection and equivalent detergent-soluble protein fractions were probed with anti-Hsp70 or anti-FLAG antibodies, or with anti-actin antibody to demonstrate equal protein loading. Over-exp; overexposed blot to highlight Hsp70-ubiquitin conjugates. (c) Parkin-mediated ubiquitination does not alter the turnover of Hsp70. SH-SY5Y cells expressing HA-tagged ubiquitin and V5-tagged Hsp70 with or without FLAG-tagged parkin were treated with cycloheximide (CHX, 100 µg/mL) at 48 h post-transfection, and cells were harvested at 0, 1, 3, 6 and 24 h thereafter. Equivalent detergent-soluble protein fractions were probed with anti-V5 and anti-FLAG antibodies to monitor Hsp70 turnover, or with anti-actin antibody to demonstrate equal protein loading. Unmodified Hsp70 is indicated by arrows and ubiquitinated forms of Hsp70 [Hsp70-(Ubn)] are also indicated. Molecular mass markers are indicated in kilodaltons (kDa). Blots are representative of at least two independent experiments.

Fig. 5. Steady-state levels and detergent-solubility of Hsp70 in parkin-linked AR-JP and sporadic PD brains

(a) Hsp70 levels are not altered in parkin-deficient AR-JP brains. Equivalent detergent-soluble and -insoluble protein fractions extracted from frontal cortex tissue of control (1–4) and parkin-deficient AR-JP brains (1–4) were analyzed by Western blot with anti-Hsp70 antibody, or anti-actin antibody as a protein loading control. Hsp70 levels were quantified by densitometry and normalized to β-actin levels in the detergent-soluble and -insoluble fraction of control (n = 4) and AR-JP (n = 4) brains. Data are expressed as a percentage (%) of control levels as the ratio of Hsp70 to β-actin, and bars represent the mean ± SEM. (b) Hsp70 displays reduced detergent-solubility in sporadic PD/DLB brains. Equivalent detergent-soluble and -insoluble protein fractions extracted from cin-gulate cortex tissue of control (5–6), Alzheimer’s disease (AD; 1–2) and PD/DLB (1–5) brains, were analyzed by Western blot with anti-Hsp70 antibody, or with anti-SOD1 and anti-β-tubulin antibodies as protein loading controls. Hsp70 levels were quantified by densitometry and normalized in the detergent-soluble and -insoluble fraction of control (control + AD; n = 4) and PD/DLB (n = 5) brains. Data are expressed as a percentage (%) of control levels as the ratio of Hsp70 to SOD1 (soluble) or β-tubulin (insoluble), and bars represent the mean ± SEM. Differences between means were analyzed by two-tailed unpaired Student’s _t_-test, *p < 0.05 when compared with control, ns, non-significant.

Fig. 6. Hsp70 and Hsc70 are multiply mono-ubiquitinated by parkin

(a) Hsp70 is mono-ubiquitinated by parkin in cultured cells. SH-SY5Y cells expressing HA-tagged ubiquitin mutants (WT, K48R or K0) and FLAG-tagged parkin were subjected to IP with anti-Hsp70 antibody, and IPs and inputs were probed with anti-HA and anti-Hsp70 antibodies to monitor Hsp70-ubiquitin conjugate formation. K48R- and K0-ubiquitin mutants fail to impair parkin-mediated Hsp70 ubiquitination. (b) Hsp70 is mono-ubiquitinated and not poly-ubiquitinated by parkin. SH-SY5Y cells expressing HA-tagged ubiquitin and V5-tagged Hsp70 with or without FLAG-tagged parkin were subjected to IP with anti-V5 antibody, and IPs and inputs were probed with ubiquitin-protein conjugate-specific antibodies that specifically recognize either poly-ubiquitinated proteins (FK1) or mono- and poly-ubiquitinated proteins (FK2). Notice only FK2 but not FK1 antibody detects ubiquitinated Hsp70. *denotes IgG heavy chain. (c) In vitro ubiquitination assay with ubiquitin mutants. Recombinant rabbit E1, human UbcH7 (E2), ba-culovirus (Bv)-derived human parkin, human Hsp70 and human ubiquitin (WT, K48R or methylated) proteins were incubated together in assay buffer containing ATP for 2 h at 37°C. Reactions were probed with anti-Hsp70 antibody to monitor Hsp70-ubiquitin conjugate formation. K48R- and methyl-ubiquitin fail to impair parkin-mediated Hsp70 ubiquitination relative to WT-ubiquitin. The absence of ubiquitin impairs Hsp70 ubiquitination. (d) Multiple mono-ubiquitination of Hsc70 by parkin. SH-SY5Y cells expressing combinations of V5-tagged Hsc70, HA-tagged ubiquitin (WT, K48R or K0) and FLAG-tagged parkin were harvested and equivalent detergent-soluble protein fractions were probed with anti-HA or anti-V5 antibodies to monitor Hsc70-ubiquitin conjugate formation. Parkin mediates Hsc70 ubiquitination that is not impaired when K48R- or K0-ubiquitin is substituted for WT-ubiquitin. Unmodified Hsp/Hsc70 is indicated by arrows and ubiquitinated forms of Hsp/Hsc70 [Hsp/Hsc70-(Ub)n] and ubiquitin-protein conjugates [(Ub)n] are indicated. Molecular mass markers are indicated in kilodaltons (kDa). Blots are representative of at least two independent experiments.

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Parkin mediates the degradation-independent ubiquitination of Hsp70 - PubMed (original) (raw)