The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to Regulate Enzyme Activity and Synaptic Vesicle Localization

Abstract

We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis.

Keywords: Hsc70; complex; dopamine; enzyme; protein targeting; synapse.

FIGURE 1.

HSC70, TH, AADC, and VMAT2 co-precipitate in MN9D cells and rat striatum. A and B, co-immunoprecipitation (IP) experiments were performed by incubating MN9D cells (A) or rat striatum (B) lysates with anti-TH, anti-AADC, or anti-Hsc70 antibodies. SDS-PAGE and immunoblot (IB) analysis using the designated antibodies demonstrated co-precipitation of TH, AADC, and Hsc70. VMAT2 also co-precipitated with these proteins in rat striatum experiments. Unrelated vesicular and cytosolic proteins (SV2, SYPH, GS, and Hsp70) failed to co-immunoprecipitate. C, similar co-immunoprecipitation experiments were performed by incubating rat striatum with the VMAT2 antibody. SDS-PAGE and IB analysis using antibodies against VMAT2, Hsc70, TH, and AADC demonstrated that these proteins co-precipitate. IB analysis with antibodies against SV2 and GAD67 showed no co-precipitation. No bands were present in any of the immunoprecipitations performed using the nonspecific IgGs or beads only.

FIGURE 2.

Specificity of the interaction between TH and HSC70. A, immunoblot analysis showing the expression of Hsc70, HSP70, GRP78, and HDP90 from MN9D cells under control conditions or after heat shock (HS). B, co-immunoprecipitation (IP) experiments were performed by incubating MN9D cell lysates with anti-TH antibody. SDS-PAGE and IB analysis using the designated antibodies demonstrated co-precipitation of Hsc70 but not HSP70, GRP78, or Hsp90. No bands were present in any of the immunoprecipitations performed using nonspecific IgGs.

FIGURE 3.

Hsc70 and TH co-localize in brain dopamine neurons. A, mouse midbrain sections of the substantia nigra (SN) and ventral tegmental area (VTA) were immunostained with polyclonal anti-TH (green, labeled with Alexa Fluor 488) and monoclonal Hsc70 (red, labeled with Alexa Fluor 555) antibodies. B, primary cell cultures from rat midbrain were immunostained with a polyclonal anti-TH (red, labeled with Alexa Fluor 555) and monoclonal Hsc70 antibodies (green, labeled with Alexa Fluor 488) antibodies. In both samples, merged images show that a majority of the TH-positive cells were dual-labeled with Hsc70 in both preparations (A and B, right panels). Only a few TH cells did not contain Hsc70. The co-localization of TH and Hsc70 extended into the process of the dopaminergic neurons. (B, right panels). Scale bars = 50 μm.

FIGURE 4.

SBD and CTD domains of Hsc70 interact with TH. A, schematic of Hsc70 and GST fusion proteins containing three functional domains: the nuclear binding domain (ATPase, residues 1–373), SBD (residues 373–540), and CBD (residues 540–650). B, even amounts of immobilized GST fusion proteins were used for pulldown experiments in MN9D cell and rat brain lysates. Samples were analyzed by SDS-PAGE and IB with antibodies against TH (first panel) and AADC (second panel). As a negative control, the pulldown samples were also analyzed using the unrelated GS antibody (third panel). Ponceau red staining (fourth panel) shows even loading of the fusion proteins. C, in vitro binding assays were performed by incubating purified His₆-TH with even amounts of the Hsc70 GST fusion proteins or GST-only prior to SDS-PAGE and IB analysis using anti-TH antibody.

FIGURE 5.

Exogenous Hsc70 increases TH activity in MN9D cells and purified synaptic vesicles. A, TH activity was measured in purified SVs obtained from mouse brain. The SVs were incubated with 10 μg of recombinant Hsc70, the closely related recombinant Hsp70 or Hsp60, or denatured Hsc70 or denatured Hsp70. The TH activity within the purified SV2 without recombinant protein (Control) was considered 100%. B, purified SVs were incubated without (Control) or with 0.1, 1, or 10 μm recombinant Hsc70. TH activity increased in a dose-dependent manner in the presence of recombinant Hsc70. C, TH activity was also assessed in the purified SVs in the presence of GST-only or GST-Hsc70 constructs. The GST-Hsc70 fusion constructs that interact with TH and AADC (GST-373–650, GST-373–540, and GST-540–650) were able to enhance TH activity in comparison with the TH activity in purified SVs alone (control, considered 100%). In contrast, non-interacting GST-Hsc70 fusion constructs (GST-1–540) did not alter TH activity. Recombinant Hsc70 was used as a positive control, and GST-only was used as a negative control. D, TH activity was assessed post-nuclear preparation from MN9D WT cells. The PNS was incubated with recombinant Hsc70, the closely related recombinant Hsp70 or Hsp60, or denatured Hsc70 or denatured Hsp70. The TH activity within the PNS alone in the control was considered 100%. *, p < 0.05.

FIGURE 6.

Hsc70 expression affects TH activity and intracellular DA concentrations in MN9D cells. A, representative IBs and densitometry analysis of MN9D cells, WT or stably transfected with scramble shRNA (Scr) or Hsc70 shRNA. Hsc70 expression was reduced in Hsc70 shRNA MN9D cells by ∼50%, whereas TH, Hsp70, and sodium/potassium ATPase expression was not significantly altered. Densitometry analysis of Hsc70 expression was normalized to arbitrary densitometry units of α-tubulin and expressed as a percentage of Hsc70 expression in MN9D WT cells. B, TH activity was examined in the PNS of these samples. The Hsc70 shRNA MN9D cells exhibited an ∼80% reduction in TH activity compared with WT and scramble shRNA stably transfected cells. C, intracellular DA levels present in the PNS of these samples were measured by HPLC. The DA levels present in the Hsc70 shRNA cells were reduced by ∼40% compared with WT and scramble shRNA MN9D cells. D, in contrast, transfection of MN9D cells with Hsc70-HA resulted in increased TH activity compared with MN9D WT cells (Control). *, p < 0.05.

FIGURE 7.

Hsc70 expression affects the subcellular distribution of TH in MN9D cells. A, an enriched SV preparation was prepared from WT, scramble shRNA, and Hsc70 shRNA cells by differential centrifugation. 50 μg of total protein from the original homogenate (H) as well as the supernatants (S) and pellets (P) from each step were analyzed by SDS-PAGE and IB using TH, SYPH, and Hsc70 antibodies. SYPH expression was enriched in the P3 sample compared with H and S3 of all three cell lines, confirming that the P3 sample represented enriched SVs. Consistent with our previous studies, TH expression was found in P3 of WT and scr-shRNA cells. Interestingly, this TH localization is shifted from P3 to S3 in Hsc70 shRNA cells. B, specific TH activity was examined in the S3 and P3 fractions of MN9D WT cells (left panel), MN9D cells expressing the scr-shRNA (center panels), and MN9D cells expressing the Hsc70 shRNA (left panels). *, p < 0.05.

FIGURE 8.

Recombinant Hsc70 is capable of restoring TH levels at the synaptic vesicle in Hsc70-shRNA cells. The synaptic vesicle fractionations were repeated under conditions where S2 was incubated alone (Control) or in combination with either recombinant Hsp70 or Hsc70 prior to obtaining S3 and P3. 50 μg of total proteins from H, S3, and P3 of WT, scramble shRNA, and Hsc70 shRNA MN9D cells were analyzed by SDS-PAGE and IB. Again, TH was enriched in P3 of WT and scramble shRNA cells in S3 of the Hsc70 shRNA cells under normal conditions. Importantly, incubation with Hsc70 restored TH levels to the P3 fraction in Hsc70 shRNA cells. Incubation with the closely related Hsp70 protein had no effect on TH distribution, confirming the specificity of the Hsc70 effect. IB analysis with SYPH, VMAT2, SV2, and secretogranin II antibodies confirmed that the fraction P3 contained synaptic vesicles.