Human Molecular Genetics Advance Access originally published online on December 7, 2007
Human Molecular Genetics 2008 17(6):895-905; doi:10.1093/hmg/ddm362
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p21-activated kinase 1 promotes soluble mutant huntingtin self-interaction and enhances toxicity
Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK
* To whom correspondence should be addressed. Tel: +44 1223762608; Fax: +44 1223331206; Email: dcr1000{at}hermes.cam.ac.uk
Received November 14, 2007; Revised November 28, 2007; Accepted December 6, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Huntington's disease (HD) is caused by a polyglutamine (polyQ) expansion in the huntingtin (htt) protein. While aggregation is a pathological hallmark of HD and related polyQ expansion diseases, the role of aggregates has been disputed. Here we report that p21-activated kinase 1 (Pak1) binds to htt in vivo and in vitro. Pak1 colocalized with mutant htt (muhtt) aggregates in cell models and in human HD brains. Pak1 overexpression enhanced the aggregation of muhtt. Furthermore, we observed SDS-soluble wild-type htt (wthtt)–wthtt, wthtt–muhtt and muhtt–muhtt interactions, which were enhanced by the presence of Pak1. We show that Pak1 overexpression enhanced htt toxicity in cell models and neurons in parallel with its ability to promote aggregation, while Pak1 knockdown suppressed both aggregation and toxicity. Overexpression of either kinase-dead or wild-type Pak enhanced both aggregation and toxicity. Our data reveal a novel mechanism regulating muhtt oligomerization and toxicity and suggest that pathology may be at least partly dependent on soluble muhtt–muhtt interactions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Huntington's disease (HD), a progressive neurodegenerative disorder characterized by abnormal movements, behavioral dysfunction and dementia (1), is caused by an expanded polyglutamine (polyQ) stretch in the huntingtin protein (htt) (2). The exact mechanism(s) of cellular toxicity caused by mutant htt remains to be elucidated. However, it is generally accepted that a toxic gain-of-function is involved. Loss-of-function mutations often show phenotypes distinct from those seen in the disease, if any at all, while disease severity appears to correlate with expression level of the mutant protein. However, some loss-of-function may contribute to the primary gain-of-function toxicity (3). The toxicity of mutant htt (muhtt) appears to be exposed after cleavage to reveal an N-terminal toxic fragment of 100–150 residues containing the expanded repeats (4–6). Such fragments are seen in the intranuclear and intracytoplasmic neuronal inclusions that characterize HD pathology (7). However, larger fragments of htt (e.g. the first 552 residues) are also seen in vivo (5).
While intracellular aggregates are seen in all the known polyQ diseases, their roles have been vigorously disputed, with some claiming that they are toxic and others arguing that they are protective or epiphenomena (8,9). While some recent data suggest that aggregates/inclusions may be protective relative to diffuse htt (9,10), it is unclear whether the toxic effects of this protein are due to soluble monomers, soluble oligomers or insoluble oligomers. Indeed, a recent study has suggested that there may be toxic monomeric conformers of polyQ expanded proteins, based on experiments with a thioredoxin model protein engineered to contain a polyQ stretch (11). Understanding this underlying principle is essential not only for HD and the eight other diseases known to be caused by polyQ expansion mutations, but also for many other neurodegenerative diseases associated with the formation of intracellular inclusions, like Alzheimer's disease and Parkinson's disease.
Wild-type htt is a 348 kDa protein, which includes multiple HEAT repeats (huntingtin, elongation factor 3, a subunit of protein phosphatase 2A and TOR1), helical motifs seen in a variety of proteins involved in intracellular transport and chromosomal segregation (12). Many proteins have been identified as htt interacting partners, providing important clues for the function of wild-type htt (wthtt) and pathology of HD (13).
p21-activated kinases (Paks) are highly conserved enzymes that are activated by small GTPases like cdc42 and Rac. Humans encode 6 Paks. The carboxy-termini of Paks1, 2 and 3 are highly conserved. Pak kinase functions overlap to some extent and include effects on the actin and tubulin cytoskeletons (14,15). Full-length Pak1, Pak2, Pak4 and Pak5 have been reported to have cytoprotective roles (16–19).
Recently, we identified cdk5 as an htt-binding protein, and found that cdk5 phosphorylates htt at Ser434 preventing htt cleavage by caspases (6). Htt can also be phosphorylated at other sites (20–22). Accordingly, we tested if other kinases or their substrates are capable of binding htt. We identified Pak1 as a novel htt-binding protein. Pak1 appears to modulate mutant htt toxicity by enhancing its oligomerization independently of its kinase activity.
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RESULTS |
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Pak1 interacts with wild-type htt
To identify the kinases or kinase substrates that bind htt, we expressed a physiologically relevant fragment, the first 552 residues of Flag-tagged wt htt (htt552) (5) in HeLa cells, then pulled down with M2 (anti-Flag antibody)-agarose. The htt552-M2-agarose complex was then incubated with mouse brain lysate and eluted with 0.1 M glycine pH3.0. Since kinases or their substrates are often phosphorylated at serines, we first blotted the eluted products with an anti-phospho-serine antibody and consistently observed a 64–68 kDa band (Fig. 1A, top panel). We then tested if a band of the same size eluted off the htt552-M2-agarose was recognized by antibodies to potential candidate proteins. Intriguingly, anti-Pak1 antibody immunoblotting detected a product of the same size as the anti-phospho-serine antibody. (Fig. 1A, middle panel). However, we detected no signals with antibodies to kinases with similar molecular weights, like Src, Yes, Fyn (data not shown). These data suggested Pak1 could be pulled down by htt552. We confirmed this result by pulling down Pak1 with wthtt552 in COS-7 cells co-transfected with Myc-Pak1 and Flag-htt552 (Fig. 1B). Wthtt588 was pulled down by Pak1 and the known htt-interactor cdk5, while Yes failed to pull down wthtt588 (Fig. 1C). We also demonstrated a physiological interaction of wt full-length htt and Pak1 in neuronal cells by immunoprecipitating htt with an anti-Pak1 antibody in mouse brain lysate (Fig. 1D). We found that the interaction between htt and Pak1 is independent of Pak1 kinase activity (Supplementary Material, Fig. S1). In Figure S1 (Supplementary Material), wild-type Pak1, kinase-dead Pak1, Pak1-299R, kinase-active Pak1 and Pak1-423E all bound similarly to htt588.
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Defining binding regions within Pak1 and htt
Next, we tried to define the domains of Pak1 that interact with htt by co-transfecting htt552 along with either empty vector control or different domains of Pak1 into HeLa cells. Full-length Pak1 (545 residues) and Pak1 residues 270–545 pulled down htt, in contrast to Pak1-1-132 and Pak1-132-270, suggesting that the C-terminal part of Pak1 bound htt (Supplementary Material, Fig. S2A). We also confirmed in COS-7 cells that Pak1-1-270 could not immunoprecipitate htt552 (Supplementary Material, Fig. S2B). The slower gel mobility of Pak1-1-270 compared with Pak1-270-545 may reflect different structures of the amino and carboxyl domains within Pak1. These data were validated in vitro using recombinant bacterially expressed GST-Pak1 deletion constructs and GST-full-length Pak1 to try to pull down in vitro-translated htt588. As in the HeLa experiments, only Pak1-270-545 and full-length Pak1 could bind to htt (Supplementary Material, Fig. S2C). Further experiments with deletion constructs suggest that the C-terminal residues 446–545 of Pak1 were sufficient to bind htt (Supplementary Material, Fig. S2D). Interestingly, Pak1-411-545, Pak1-436-545, Pak1-441-545, Pak1-446-545 show dimer-like pattern (*labeling, Supplementary Material, Fig. S2D), consistent with its ability to dimerize (14). Figure 1E shows htt552 can be pulled down by Pak1, Pak1-270-545 or Pak1-446-545 using anti-Myc antibody, confirming that the Pak-C-terminal binds to htt. Using the same approaches we tried to map the Pak1-binding region of htt. Htt120 and htt190 bound Pak1 weakly, while stronger interactions were seen between Pak1 and htt315 or htt588 (Fig. 1F).
Pak1 interacts with mutant htt
We demonstrated that Pak1 interacted with polyQ-expanded htt (muhtt) by cotransfecting either wthtt588 or muhtt588 (both Flag-tagged) along with Myc-tagged Pak1 into COS-7 cells. After immunoprecipitating with anti-Myc (Supplementary Material, Fig. S3A), muhtt was detected, and after immunoprecipitating with anti-Flag we detected the Pak1 (Fig. 2A). The input versus immunoprecipitate ratio for wthtt588/Pak1 compared with muhtt588/Pak1 (Fig. 2A, Supplementary Material, Fig. S3A) suggested that wthtt588 may interact more strongly with Pak1. We confirmed that muhtt binds to the same domain of Pak1 as wthtt, by showing that full-length Pak1 and Pak1 residues 270-545 pulled down htt, rather than Pak1-1-132 or Pak1-132-270 (Fig. 2B). Since muhtt exon1 (muhttEx1) is often used in cell and mouse models of HD, we tested whether muhttEx1 was able to bind to Pak1. Figure 2C shows muhttEx1 does not bind to Pak1. Figure S4 (Supplementary Material) summarizes the Pak1 variants used for htt binding assays and htt fragments used for Pak1 binding assays and the relevant interactions.
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Pak1 enhances muhtt aggregation
Since cdk5 phosphorylates muhtt and affects aggregation (6), we initially tested whether kinase-active Pak1 modulated muhtt aggregation. A constitutively active form of Pak1, Pak1-423E, enhanced muhtt aggregation (Fig. 3A). We next tested whether this effect was due to Pak1 binding to htt or Pak1 phosphorylation of htt because we found that Pak1 binding to htt was independent of kinase activity (Supplementary Material, Fig. S1). So, we evaluated the effects of the expression of wt Pak1, kinase-dead Pak1 (Pak1-299R), or constitutively active Pak1 on muhtt aggregation, and found that all three forms enhanced aggregate formation (Fig. 3B). These data suggested muhtt aggregation is influenced by Pak1 binding rather than kinase activity. To confirm this hypothesis, we tested whether Pak1 affected aggregation of exon1-74Q of htt (httEx1-74Q), since muhttEx1 does not bind Pak1 (Fig. 2C). HttEx1-74Q aggregation was not affected by Pak1 overexpression, suggesting that these effects of Pak1 on htt aggregation required binding to htt (Fig. 3C). Likewise, aggregation was only increased by forms of Pak1 that bound htt (Fig. 3D). The effects of Pak1 on htt aggregation were not due to alterations in soluble htt levels (Supplementary Material, Fig. S5A). Pak1 colocalized with htt aggregates in SK-N-SH neuroblastoma cells, in cultured cortical and striatal neurons and in HD patient brains (Fig. 4A–D). All (100%) of cortical or striatal neurons with such inclusions had aggregates containing both htt and Pak1. Almost 100% (>95%) of aggregates in HD brains were decorated with Pak1 antibodies (determined using double staining as in Fig. 4C).
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Pak1 promotes htt–htt interaction
Aggregate formation requires protein–protein interactions, so we tested if htt interacted with itself using cells transfected with Myc- and Flag-tagged wthtt588. Figure 5A shows that Myc-htt588 was pulled down with Flag-htt588. Likewise, Myc-htt588 could pull down untagged wthtt548 (Supplementary Material, Fig. S5B). Consistent with the above data, Myc-Pak1 also pulled down untagged wthtt548, while Myc-Beclin 1 (negative control) failed to pull down untagged wthtt548. To map the domain of htt that is essential for this self-interaction, we transfected different Flag-htt variants with Myc-htt588 into HeLa cells and showed that htt315 was sufficient for the self-association (Supplementary Material, Fig. S5C). Htt190 bound only weakly to htt588 and htt120 appeared to bind htt588 even more weakly (Supplementary Material, Fig. S5C). Neither deletion of the polyP nor the polyQ tracts found in exon1 abolished the interactions with htt588 (Supplementary Material, Fig. S5D).
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We then tested whether Pak1 regulated htt–htt interactions, since Pak1 can increase muhtt aggregation. Myc-htt588 and Flag-htt588 were transfected into HeLa cells with increasing amounts of Pak1 (but keeping total amount of transfected DNA equivalent). Figure 5B shows that increasing amounts of Myc-htt588 were pulled down by Flag-htt588 as the amount of Pak1 was increased.
Next, we confirmed that the Pak1 effect on wthtt–wthtt interaction was also seen with muhtt–wthtt (Fig. 5C) or muhtt–muhtt (Fig. 5D) interactions. The muhtt–muhtt (and wthtt) interactions are SDS-dissociable, as the pulled-down products migrate like monomers on reducing gels. Thus, these interactions that are enhanced by Pak1 are weaker than those in the SDS-resistant aggregates formed by muhtt (23) that also do not enter the resolving gel (24). Such SDS-soluble muhtt–muhtt interactions may be oligomeric precursors of aggregates/inclusions, and thus these data can explain why Pak1 can enhance muhtt aggregation.
Cell death is aggravated with increased oligomerization
Next, we tested if the increase in muhtt aggregation/oligomerization mediated by Pak1 was associated with increased cell death. In HeLa cells, as expected, aggregation did not change with Pak1-N (Pak1-1-132), the non-htt binding domain of Pak1, but dramatically increased in the presence of the htt-binding domain, Pak1-C (Pak1-270-545), wt-Pak1, Pak1-299R and Pak1-423E (Fig. 6A). The proportion of cells showing apoptotic morphologies correlated with aggregation, and was significantly elevated in the cells overexpressing forms of Pak1 that enhanced muhtt aggregation (Fig. 6A). Note that Pak1 overexpression does not enhance cell death on its own (Supplementary Material, Fig. S6A), and muhtt548-GFP is significantly more toxic than wthtt548 (which is no more toxic than the empty vector control; Supplementary Material, Fig. S6B and C). Consistent with the HeLa cell data, overexpression of Pak1-1-270 (which does not bind htt) neither altered htt aggregation nor cell death, while both Pak1 and Pak1-270-545 dramatically increased aggregation and cell death in SK-N-SH neuroblastoma cells (Fig. 6B). However, Pak1 overexpression did not appear to alter the cellular localization of muhtt aggregates (Supplementary Material, Fig. S6D). Figure S7 (Supplementary Material) shows an example of muhtt548-GFP aggregation and cell death associated with Pak1 overexpression.
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We confirmed the effects of Pak1 on muhtt aggregation and toxicity in primary cortical neurons which are affected early in HD (25). In these neurons, mutant htt is diffusely distributed when there are no overt inclusions. However, the muhtt appears to be largely sequestered in inclusions after they form. Consistent with our data in cell lines, Pak1 overexpression dramatically increased the proportion of htt-expressing cells with inclusions and apoptotic morphology (Fig. 7A). As we observed in the cell lines, similar effects were mediated by kinase-dead Pak1-299R as with wt-Pak1, suggesting these effects were kinase activity-independent (Fig. 7A). However, no toxicity was observed with Pak1 or Pak1-299R alone in these neurons (data not shown). As the striatum is affected very early in HD, we also examined these effects in rat striatal neurons, where Pak1 also significantly increased the proportion of muhtt-expressing cells with inclusions and muhtt-mediated toxicity (Fig. 7B). Figure 7C shows examples of aggregates and nuclei in muhtt548-GFP/Pak1 transfected striatal neurons.
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Pak1 knockdown reduces mutant htt aggregation and cell death
Knockdown of endogenous Pak1 expression reduced both muhtt aggregation and cell death (Fig. 8). These data suggest that endogenous Pak1 in its physiological context also influences muhtt aggregation and toxicity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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We have identified Pak1 as an htt interactor that modifies muhtt toxicity. This is most likely to be directly due to its ability to bind to htt and mediate soluble muhtt–muhtt interactions. The domains of Pak1 that bind htt facilitate oligomerization/aggregation, and no enhanced toxicity was observed with Pak1 domains that do not bind htt. Importantly, Pak1 also enhances oligomerization of wt htt (that does not form any aggregates). This suggests that Pak1 plays a key role in enhancing htt–htt interactions in a way that is independent from effects of the ‘sticky’ expanded polyQ tract. As kinase-dead Pak1 overexpression enhanced aggregation and toxicity, the effects were not a function of Pak1 kinase activity, but due to Pak1 physical interactions. While most previous studies investigating the role of htt aggregation and toxicity have used exon 1 fragments (which may not exactly represent what are seen in vivo, and which do not interact with Pak1) or model proteins like thioredoxin with expanded polyQs (11), we have used longer fragments of htt. (Full-length htt is not practical for the cell-based experiments as the construct is very large (>15 kb), mitigating against efficient transfection and is associated with very low rates of aggregation and toxicity, making such experiments unreliable.).
Previous studies have reported that htt-binding peptides (26), htt-binding intrabodies (27), chemical (8) and molecular chaperones (28) reduce aggregation/inclusion formation and toxicity of mutant htt fragments. One possible explanation that may be used to reconcile these data with those suggesting that inclusions are less toxic than diffuse mutant htt, was that such htt-binding peptides or compounds that reduce aggregation are protecting against toxicity by shielding a reactive epitope that may cause toxicity via a mechanism independent of aggregation. One could argue that those mutant htt interactors prevented aggregation as an epiphenomenon, as the same regions of htt that mediated aggregation were ‘sticky’ and toxic, leading to abnormal binding to and malfunction of other proteins. Most studies that have been performed to date addressing this issue have used less than the first 70 amino acids of htt, and thus comprise mainly polyQ, which is known to aggregate rapidly. Indeed, one could extend the argument to propose that the aggregation actually results in the loss of such toxic epitopes, and is thus protective.
We believe that our data can be reconciled with much of the previous apparently contradictory data relating to htt aggregates and toxicity. If the dynamic process of htt–htt interaction, or the presence of specific oligomeric structures are the toxic entities, then agents that block the formation of such early structures like htt-binding intrabodies, and chaperones will have protective effects and will also reduce the formation of the large aggregates visible by light microscopy as an epiphenomenon, since the formation of aggregates will be driven by the ability of the protein to form lower-order oligomers. Likewise, the conversion of such SDS-soluble muhtt dimers/oligomers to large insoluble inclusions will deplete the pool of potentially toxic oligomers and monomers with the potential for oligomerization, thus explaining why cells with visible inclusions survive longer than cells with diffusely distributed muhtt (which may be oligomeric, since wthtt also oligomerizes) (8). However, our data do not preclude the possibility of toxic monomeric conformers (11), but do argue that oligomeric forms may be more toxic.
Our identification of Pak1 as the first known direct htt interactor that enhances SDS-soluble muhtt–muhtt interactions has provided a tool to study the relationship between muhtt oligomerization and toxicity, as it bypasses the argument used for chaperones, intrabodies, etc. that they are blocking toxic epitopes. This caveat is further minimized as Pak1 binds quite far away from the N-terminal polyQ stretch. As Pak1 is mediating htt–htt interactions at a very early stage even with wthtt in a manner independent of its kinase activity, this argues that early steps in the htt oligomerization cascade are likely to be toxic, and oligomerization and/or oligomeric species are likely to be more toxic than monomers. Indeed, this is likely to be regulated at very early stages in the aggregation process, as Pak1 is facilitating muhtt–muhtt interactions that are soluble, long before insoluble aggregates form. Our data vindicate the search for compounds that block this process as a therapeutic strategy. However, many of the large-scale attempts to identify aggregation inhibitors for HD have focused on exon 1 or isolated polyQ htt fragments (29,30). The identification of the Pak1 interaction as lying outside exon1 and the fact that it modulates interactions of longer htt fragments but not exon1 fragments suggests that it may also be worthwhile considering establishing high-throughput assays for inhibitors with longer htt fragments, as these may be associated with a greater number of tractable targets.
In conclusion, the functional links between htt and Paks may provide clues for the treatment of HD. One may be able to design (or screen for) small molecules that can prevent Pak1-mediated htt oligomerization.
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MATERIALS AND METHODS |
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Vectors, cells and DNA construction
Htt548-GFP-138Q and htt548-138Q were provided by Dr M.R. Hayden (University of British Columbia, Vancouver, British Columbia, Canada); wild-type Pak1 and Pak1-423E were provided by Dr J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA); GFP-Pak1 siRNA construct (Pak1 pSUPER-GFP) was a kind gift from Drs D. Wu and Z. Li (University of Connecticut Health Center, Farmington, CT) (31). Mouse htt–/– and control ES cells were kindly provided by Dr Marcy MacDonald (Harvard). Flag-tagged N-terminal htt constructs: htt552 (amino acids 1–552), htt315 (amino acids 1–315), htt190 (amino acids 1–190), htt120 (amino acids 1–120) were generated by PCR and subcloned into pCI vector flanked by Mlu I and Not I. Other htt constructs were generated as described previously (6). Pak1-299R was generated by Stratagene Quikchange Kit. GST-Pak1 or variants were cloned into pEGX-6P (Pharmacia Biotech). All the constructs have been confirmed by DNA sequencing.
Antibodies and reagents
Rabbit polyclonal antibodies: anti-Pak1 (N-20) (1:1000); anti-Myc (1:1000) (Sigma), c-Yes (1:1000) (upstate, Santa Cruz). Goat polyclonal antibody: anti-Pak1 (1:500) (Santa Cruz, CA). Anti-mouse monoclonal antibodies: anti-htt (MAB2166) (1:1000), anti-htt (EM48) [Chemicon International (Temecula, CA)], anti-Flag (M2) (1:1000), anti-tubulin (1:5000) (Sigma), anti-phosphoserine, 16B4 (Calbiochem), anti-cdk5 (J3) (1:1000) (Santa Cruz). Anti-Flag M2-agarose affinity gel, anti-mouse or rabbit IgG agarose (Sigma). TNT-coupled reticulocyte lysate (Promega); and 35S-methionine (Amersham, UK).
Cell culture
COS-7, HeLa, SK-N-SH and MCF-7 cells were cultured with standard methods in DMEM supplemented with 10% FCS (Sigma). Transfection was performed with Lipofectamine or Lipofectamine Plus (Invitrogen) according to standard methods. Rat E18 cortical and striatal neurons were purchased from Neuromics (MN) or prepared from E18 rat brain tissues according to instructions from Neuromics, cultured in neurobasal (Invitrogen) medium supplemented with B27 (Invitrogen) and 0.5 mM glutamine and transfected with Lipofectamine 2000 according to standard methods.
Immunohistochemistry
We used paraffin-embedded brain slices from humans with grade III HD and unaffected humans, spanning the caudate and putamen regions. We carried out double immunohistochemical staining using PicTure-Double Staining Kit (Zymed Laboratories, CA) or single immunohistochemical analysis by standard peroxidase labeling using the Vectastain Avidin: Biotinylated enzyme Complex (ABC) kit. We followed experimental instructions from the PicTure-double staining kit. Primary antibodies were incubated overnight. Pictures were taken under Zeiss Axioskop microscope (Carl Zeiss, Welwyn Garden City, UK), 63x 1.4NA Plan Apochnomat oil immersion, room temp, AxioVision LE, Rel 4.2 software. Local Ethical Committee approval has been given for the use of HD and control samples.
Immunocytochemistry
After two washes with phosphate-buffered saline (PBS), cells were fixed with 4% paraformadehyde for 10 min. The fixed cells were washed three times in PBS, then permeablized with 0.5% Triton in PBS for 10 min. Cells were blocked in blocking buffer (1% BSA, 1% heat inactivated goat serum in PBS) for 30 min at room temperature. Primary antibodies were incubated with cells overnight at 4°C. The secondary antibody was incubated for 30 min after washing three times (10 min, each). Cells were washed three times (10 min, each) after incubation with secondary antibodies, then mounted with DAPI (3 µg/ml). Images were acquired on an LSM510 META microscope (Carl Zeiss, Welwyn Garden City, UK), 63x 1.4NA Plan Apochnomat oil immersion, at room temperature and using Zeiss LSM510 v3.2 software.
Immunoprecipitation
Immunoprecipitation (IP) was performed using Buffer A [20 mM Tris–HCl, pH 7.2, 2 mM MgCl2, 150 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 0.5% NP-40, protease inhibitor cocktail (Roche)]. Cells were lysed in Buffer A for 20 min on ice, followed by centrifugation at 13 000g for 15 min. Five hundred micrograms to one milligram total protein were used as the starting material for IPs [protein concentrations were determined using a Bio-Rad (Hercules, CA) protein assay and spectrophotometer with BSA standard curve according to manufacturer's instructions]. Primary antibodies (or anti-Flag M2-agarose affinity gel) were added to a final concentration of 5 µg/ml and incubated for 2 h to overnight at 4°C. Anti-mouse or rabbit IgG agarose were added to the mixture and incubated at 4°C for 1 h. Following three washings, IP products were either directly boiled in Laemmli buffer or eluted with 0.1M glycine (pH 2.3) then boiled in Laemmli buffer, and subjected to PVDF membrane transfer and western blot.
We identified Pak1 as follows. One 10 cm dish COS-7 cells was transfected with Flag-htt552. The cell lysate was incubated with M2-agarose affinity gel for 2 h. After one wash, the M2 gel was then incubated with 5 mg mouse brain total protein overnight. The IP products were eluted with 0.1 M glycine (pH 2.3) and subjected to western blot.
Estimation of cell death and aggregates
To measure cell death or aggregates, approximately 200 transfected cells (approximately 100 transfected cortical and striatal neurons) were counted in multiple random visual fields per slide. All coverslips were scored with the observer blinded to the identity of the slides. Cells were analysed using a fluorescent microscope (Eclipse E600, Nikon, Japan). The figures show data from representative experiments in triplicate. Cell death was monitored by scoring the transfected cells with apoptotic nuclear morphology—fragmented or pyknotic nuclei. Cells were counted as aggregate-positive if one or several aggregates were visible within a cell. P-values were determined by unconditional logistical regression analysis by using the general loglinear option of SPSS 9.1 software (SPSS, Chicago, IL).
GST pull-down assay
GST or GST-Pak variants expressed in BL21 (DE3) were adsorbed to glutathione–agarose beads for 1 h, followed by three PBS washes. The bound GST or GST-Pak variants were incubated with in-vitro translated htt588 for 2 h at 4°C, then washed three times in Buffer A. The bound proteins were resolved by SDS–PAGE and dried. Films were exposed with the dried gel.
In vitro translation and cleavage
In vitro translations were performed in TNT-coupled reticulocyte lysate systems following Promega instruction.
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SUPPLEMENTARY MATERIAL |
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Supplementary Material is available at HMG Online.
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FUNDING |
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We are grateful to the MRC and the Wellcome Trust (Senior Clinical Fellowship) for funding to D.C.R and his group. Funding to pay Open Access publication Charges for this article was provided by the Wellcome Trust.
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ACKNOWLEDGEMENTS |
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We thank Dr M.R. Hayden for muhtt548-GFP and muhtt548, Drs J. Chernoff for Pak and Pak and Pak423E, Drs D. Wu and Z. Li for Pak1 pSUPER-GFP construct and the Institute of Psychiatry (London) Brain Bank for HD samples. We are grateful to Cahir O'Kane, Jessica Lam and Sara Imasio for helpful discussion.
Conflict of Interest statement. None declared.
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FOOTNOTES |
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Present address: Department of Neurology, Saga University, Nabeshima, Saga 849 8501, Japan. 
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