Htt interacts with cdk5
The overlapping subcellular localizations and functions of htt and cdk5 and the presence of multiple minimum cdk5 phosphorylation sites within htt led us to test if these proteins interact. Residues 1–588 of htt (htt588) pull down cdk5 ( A) in COS-7 cells. In the middle panel of A, cleavage products of htt588 are seen, which likely correspond to htt552 and htt513 (Wellington et al., 2000
). Using the same coimmunoprecipitation approach, we showed that httwt588 (aa 1–588 of wild-type htt with 17 glutamines-17Q), wtFLhtt (wild-type, full-length htt with 17Q), httmu588 (aa 1–588 of mutant htt with 138Q), and muFLhtt (mutant full-length htt with 138Q) all bound cdk5 ( B). To narrow down the cdk5-binding region of htt, we expressed GST fused to amino acids 5–56 of htt (GST-htt5-56) in Escherichia coli
, pulled down the recombinant protein with glutathione-sepharose, and incubated it with cdk5-transfected COS-7 cell lysate. C shows that residues 5–56 of htt bind cdk5. We confirmed the physical interaction of htt and cdk5 in neuronal cells by immunoprecipitating endogenous htt in mouse brain lysate with anti-htt and demonstrating that cdk5 was also pulled down with htt ( D, i). D (ii) shows the reverse experiment and confirms that endogenous htt can also be immunoprecipitated by anti-cdk5. We could not detect any binding between the cdk5 activator, p35, and htt (unpublished data). These data reveal a selective physical interaction between htt and cdk5.
Figure 1. Htt interacts with Cdk5. (A) COS-7 cells were cotransfected with empty vector and cdk5 (lanes 1–3) or Flag-tagged htt1-588 (htt588) and cdk5 (lane 4). In all experiments, empty vector controls have been performed where appropriate and the amount (more ...)
Htt associates with cdk5 in membrane fraction
Htt is enriched in membrane fractions (Gutekunst et al., 1995
). To further investigate if htt and cdk5 interact significantly, we tested if overexpression of htt led to an enrichment of cdk5 in light membranes (LM) such as endosomes and ER vesicles. Cdk5/empty vector or cdk5/htt551 were transfected to COS-7 cells. A shows similar total cdk5 levels in both cdk5-transfected cells and cdk5/htt-transfected cells (actin, enriched in LM, was used as a protein loading control for total and LM lysates). However, cdk5 levels in LM were much higher in cells where cdk5 (rather than empty vector) was cotransfected with htt. We tested the converse prediction that immunodepletion of htt would reduce cdk5 levels in LM using endogenous htt and cdk5 in mouse brain lysates. In both cytosolic and particularly in LM fractions, cdk5 levels were reduced after htt immunodepletion with anti-htt (181-500 aa; as a function of actin levels; B). Thus, the reduction of the cdk5 level is a specific consequence of htt immunodepletion, further confirming the interaction between htt and cdk5 in LM. In neuronally differentiated rat pheochromocytoma cells (PC-12 cells), we confirmed colocalization of endogenous htt and cdk5 ( C).
Figure 2. Htt associates with cdk5 at LM. (A) Cdk5 was cotransfected with empty vector/cdk5 (2:1) or Flag-htt1-551 (htt551)/cdk5 (2:1) in COS-7 cells. After 24 h, transfected cells were harvested. Light membranes (LM) (including endosomes and all ER vesicles) were (more ...)
Cdk5 phosphorylates htt in vitro and in vivo
The htt–cdk5 interaction suggested that we should test if htt was a cdk5 substrate. Six minimal cdk5 phosphorylation sites, comprising Ser-Pro or Thr-Pro, were found within htt588. GST-htt588 (but not the control protein, GST) is readily phosphorylated by recombinant p35–cdk5 complex in vitro. ( A). Next, we immunoprecipitated p35–cdk5 complexes from transfected COS-7 cells and incubated these complexes with either httwt588 or httmu588 immunoprecipitated from COS-7 cells. In vitro phosphorylation assays showed that p35–cdk5 could phosphorylate both httwt588 and httmu588 to similar extents ( B). In PC-12 cells, cdk5 activity dramatically increases after NGF differentiation (Harada et al., 2001
). To test if htt is phosphorylated by cdk5 in vivo, we differentiated PC-12 cells with NGF and treated cells with the cdk5 inhibitor roscovitine or DMSO, and then pulled down the endogenous htt with anti-htt. Serine phosphorylation of htt was assessed with the antiphosphoserine antibody 16B4. As C shows, htt phosphorylation can be detected after differentiation but was abolished with roscovitine (which inhibits cdk5). In vitro kinase assays using histone H1 as a substrate were used to confirm that roscovitine is a potent inhibitor of cdk5 under these conditions ( C).
Figure 3. Cdk5 phosphorylates htt in vitro and in vivo. (A) GST and GST-tagged htt1-588 (GST-htt588) (wild-type) were purified from E. coli. Both proteins were phosphorylated by 0.1 μg of p35–cdk5 complexes. Top panel shows phosphorylated GST (lane (more ...)
Cdk5 phosphorylates htt at Ser 434 in vitro and in vivo
We mapped the cdk5 phosphorylation site(s) by first testing p35–cdk5 phosphorylation of different truncated htt fragments in vitro. A (i) suggests the phosphorylation site(s) may be between aa 551 and 415, as phosphorylation was observed with htt551 but not with htt415 or htt314. We mutagenized the potential cdk5 phosphorylation sites within this region. When Ser 434 (S434) was mutated to Ala, cdk5 failed to phosphorylate htt588 in vitro ( A, ii), suggesting that S434 is the only cdk5 phosphorylation site in htt588. We then tested if htt S434 was phosphorylated in vivo. Htt551/empty vector, htt551/p35, and htt551 S434A/p35 were cotransfected into HeLa cells, which express cdk5 but not the activators like p35/p39. After 32P metabolic labeling, htt551 was phosphorylated only in the presence of p35 (a cdk5-specific activator), confirming the specificity of the reaction to cdk5 kinase activity. Consistent with this specificity, the phosphorylation of htt was inhibited by roscovitine. Mutation of htt S434 to A also prevented phosphorylation in vivo, which is consistent with our in vitro data suggesting that this was the phosphorylation site in vivo ( B). This consensus cdk5 phosphorylation site is conserved in htt orthologues across many vertebrates ( C).
Figure 4. Cdk5 phosphorylates htt at Ser434 in vitro and in vivo. (A, i) htt1-551 (htt551), htt1-415 (htt415), and htt1-314 (htt314) were immunoprecipitated with anti-Flag from COS-7 cells, and then γ-[32P]ATP and 0.1 μg of recombinant p35–cdk5 (more ...)
To further confirm that htt is phosphorylated by cdk5 in vivo, we developed an anti–htt-pS434 antibody (anti-pS434; see Materials and methods). To test the antibody, we first phosphorylated either GST or GST-htt588 using p35–cdk5 complex in vitro, and then probed gels of the reaction products with anti-pS434. Anti-pS434 detected phospho–GST-htt588 (rather than GST) that had an electrophoretic mobility similar to the GST-htt588; comparable amounts of GST and GST-htt588 were confirmed by anti-GST probing ( D). As htt can be phosphorylated at S434 in HeLa cells in the presence of cdk5 activity ( B), we tested S434 phosphorylation in HeLa cells using the anti-pS434 antibody. When htt551 or htt551 S434A were cotransfected with either empty vector or p35 into HeLa cells, anti-pS434 only detected htt phosphorylation when cdk5 was activated (in the presence of p35) and required S at residue 434, consistent with B ( E). F shows that mutant polyQ-expanded htt551 (muhtt551) was phosphorylated at S434 in HeLa cells in the presence of, but not the absence of, p35. Thus, anti-pS434 recognizes phosphorylated htt and only recognizes the specific band when htt is phosphorylated at residue 434. To further show that htt is phosphorylated by cdk5 in vivo, we first transfected PC-12 cells with empty vector ( G, lane 1), cdk5 ( G, lane 2), and cdk5 dominant-negative form (cdk5DN; G, lane 3) or treated with the cdk5 inhibitor roscovitine ( G, lane 4). The cells were then starved for 24 h and induced with NGF, and transfected cells were sorted with FACS. In cdk5-transfected cells, phosphorylation of endogenous full-length htt slightly increased, but in cdk5DN transfected cells, the phosphorylation is markedly reduced compared with empty vector transfected cells, and in the roscovitine-treated cells, the phosphorylation is fully disabled ( G). These data also confirm that anti-pS434 can specifically recognize cdk5-phosphorylated forms of full-length htt. Anti-pS434 identified a band of the same mobility as the roscovitine/cdk5DN-sensitive band seen in the PC-12 cells in newborn mouse brain, suggesting that endogenous mouse htt is phosphorylated at S434 ( G, lane 5).
Cdk5 phosphorylation of mutant htt reduces its toxicity
To investigate if cdk5 could modulate mutant htt toxicity, we transfected httmu588 (httmu588 [138Q] with empty vector), httmu588/p35 (httmu588 with p35), httmu588 S434A (httmu588 S434A with empty vector), or httmu588 S434A/p35 (httmu588 S434A with p35) into SK-N-SH (neuroblastoma) cells, which express cdk5 but not the activators like p35/p39 ( A). Activation of endogenous cdk5 kinase activity in these cells by p35 reduced the proportions of cells with aggregates or cell death in the cells expressing httmu588 but did not reduce either the proportions of cells with aggregates or cell death in cells expressing httmu588 S434A, the nonphosphorylatable httmu588 form ( A). Thus, activation (by p35) of cdk5 activity is required in order for it to protect against the toxicity of httmu588 in neuronal cells. Furthermore, this is not a nonspecific protective effect of cdk5 activation but requires S at residue 434 in httmu588.
Figure 5. Cdk5-phosphorylating htt blocks caspase cleavage and regulates mutant htt toxicity. (A) Httmu588/vector, httmu588/p35, httmu588 S434A/vector, and httmu588 S434A/p35 were transfected to cdk5-expressing neuroblastoma SK-N-SH cells. After 48 h, cells were (more ...)
Cdk5 phosphorylation of htt reduces its cleavage by caspases
We considered that p35–cdk5 may protect against httmu588 by modulating its turnover, but this hypothesis was not supported by our initial experiments (unpublished data). Because cdk5 phosphorylation of httmu551 was associated with a reduction of its cleavage product htt 513 ( F), we tested if p35–cdk5 phosphorylation modulated htt cleavage. Because htt cleavage at sites close to S434 is mediated by caspases, we established assays for htt cleavage in HeLa cells treated with low doses of the caspase-inducing drug staurosporine. We transfected p35 into HeLa cells to activate cdk5 kinase activity. B shows that staurosporine treatment results in cleavage of htt551 (e.g., B, lanes 5 and 6). The cleavage of htt551 (as judged by the ratio of htt551 to 513) in the cells with p35 transfection ( B, lane 2) was obviously less than that in cells without p35 transfection ( B, lane 6). Also, p35 expression does not alter htt551 expression (unpublished data). Although p35–cdk5 activity regulated htt551 cleavage induced by staurosporine, it did not reduce staurosporine-induced cleavage of the htt551 S434A mutant ( B, lanes 4 and 8). Thus, htt551 cleavage is specifically regulated by p35–cdk5 acting at S434. Some htt551 cleavage occurred in the absence of staurosporine and p35 ( B). This result may be due to low levels of active caspases (e.g., resulting from transfection), although we cannot exclude a role for other proteases.
We tested if p35–cdk5 regulated cleavage of mutant htt. C shows that httmu551 cleavage ( C, lanes 1 and 2) was consistently much less in p35-expressing cells, compared with cells not transfected with p35 ( C, lanes 3 and 4) in either staurosporine-treated or untreated conditions. Staurosporine-induced htt551 cleavage in cell models is caspase-dependent, as cleavage of htt551 was effectively prevented by the pan-caspase inhibitor Z-VAD-fmk (unpublished data). Because mutant htt cleavage enhances its aggregate formation and toxicity, these data can account for the protective effects observed against httmu588 by p35–cdk5 ( A).
Because Wellington et al. (1998)
) reported that htt can be cleaved by caspase-3 at D513, we tested if cdk5 phosphorylation of htt changed its susceptibility to caspase-3 cleavage in vitro. In vitro–translated 35
S-labeled htt551 was phosphorylated in vitro in the presence of recombinant p35–cdk5 and ATP-γ-S, which can phosphorylate htt551. The use of ATP-γ-S ensures stability of the phosphorylated site, as the resulting thiophosphorylation is resistant to phosphatase attack. Cdk5-phosphorylated htt551 ( D, lane 3) is cleaved less by caspase-3 compared with htt551 that has not been cdk5 phosphorylated ( D, lane 2) or htt551 S434A with or without cdk5–p35 phosphorylation (showing the effect is dependent on the presence of htt S434; D). Quantification of D revealed that cdk5 phosphorylation of htt551 increased the ratio of htt551 to htt513 by 1.7-fold in the presence of caspase-3, whereas there was no obvious effect of this phosphorylation in the htt551 S434A mutant (under the same conditions). The phosphorylation probably does not have a more pronounced effect in this experiment, as it is very likely that only a proportion of the molecules are stably phosphorylated (and thus protected from cleavage) under in vitro conditions. Thus, cdk5 phosphorylation of htt prevents its cleavage by caspases and can account for activated cdk5 inhibiting httmu588-induced cell death and aggregation. Because htt phosphorylation by cdk5 prevented its cleavage by caspases in vitro, it is likely that this effect is regulated by phosphorylation itself rather than by interaction of other proteins at the phosphorylation site.
To confirm that cdk5 activity is an important regulator of full-length htt cleavage in vivo, we studied neuronally differentiated PC-12 cells (Sugars et al., 2004
), which express either NH2
-terminal Flag-tagged wild-type or mutant, full-length htt under the control of a doxycycline-responsive promoter (Tet-On). We can detect NH2
-terminal cleavage products of these transgenes specifically using an anti-Flag antibody in these cell lines. The transgene expression levels in these cells is similar to the endogenous htt (Sugars et al., 2004
). We used these cells to test our prediction that inhibition of cdk5 activity with its inhibitor roscovitine would result in more transgene cleavage at residue 513. Compared with untreated cells, roscovitine-treated NGF-differentiated cells produced more of the caspase cleavage fragment, htt513 ( A). The ratio of htt552 to htt 513 was dramatically reduced in the roscovitine-treated cells, which is consistent with greater cleavage at amino acid 513 in the absence of cdk5 activity. In stable inducible, neuronally differentiated PC-12 cells expressing mutant 138Q full-length transgenes, enhanced htt cleavage would be predicted to result in more aggregates, as aggregation is enhanced by short NH2
-terminal htt products, compared with longer fragments. Consistent with our previous data showing that cdk5-mediated phosphorylation reduces htt cleavage at D513, aggregate formation was substantially increased in neuronally differentiated PC-12 cells treated with roscovitine while expressing the transgene, compared with untreated cells ( B, i and ii). Compared with untreated cells, roscovitine treatment of these cells produced more of the caspase-cleavage fragment htt513 ( B), and the ratio of htt552 to htt 513 was dramatically reduced in the roscovitine-treated cells, which is consistent with greater cleavage at amino acid 513 in the absence of cdk5 activity. Roscovitine was not toxic to PC-12 cells under these conditions (unpublished data). We could not evaluate mutant htt-induced cell death in this model as cell death rates are very low in this cell model expressing mutant full-length htt (Sugars et al., 2004
Figure 6. Inhibition of cdk5 activity promotes htt cleavage, and mutant htt aggregate formation in differentiated htt stable PC-12 cells. (A) Wild-type, full-length htt PC-12 cells were differentiated with 100 ng/ml NGF for 48 h. 1 μg/ml doxycycline was (more ...)
Cdk5 activity is reduced in HD transgenic mouse brains
Because cdk5 activity is protective against mutant htt cleavage and toxicity, we investigated if this activity was altered in a mouse model of HD expressing the first 171 residues of htt with expanded polyQs (Schilling et al., 1999
). Surprisingly, the specific activity of cdk5 immunoprecipitated from brains of mice carrying the mutant transgene was significantly lower than that (of comparable amounts of cdk5) from wild-type littermates ( A). However, p35 levels were not reduced in brain lysates from the mutant HD mice ( B). Furthermore, cdk5 and p35 were not sequestered into either cytosolic or nuclear htt aggregates in vivo. Samples from human HD brains and HD transgenic mouse brains (and relevant non-HD controls) were analyzed using both peroxidase and immunofluorescence detection systems and no aggregates staining for either cdk5 or p35 were seen, whereas convincing aggregate staining was seen with both antiubiquitin and anti-htt antibodies (unpublished data).
Figure 7. Mutant htt impairs cdk5 activity by interfering p35–cdk5 interaction. (A) Cdk5 was pulled down from the lysates of wild-type littermate control (Ctrl; lane 1) or HD (lane 2) mice whole brains (12 wk). Histone H1 was used as a substrate (more ...)
Because htt interacts with cdk5, we hypothesized that the polyQ expansion may reduce cdk5 activity by impairing its interaction with p35. C shows that much less p35 was pulled down by anti-cdk5 from HD compared with wild-type littermate brain lysate, although comparable amounts of cdk5 were pulled down in the two samples, supporting our hypothesis that mutant htt impairs the p35–cdk5 interaction. Next, we used GFP-htt exon1-23Q (GFP-httEx1-23Q) and GFP-htt exon1-74Q (GFP-httEx1-74Q) in cellular models to confirm the data from HD mice because htt exon1 is sufficient to bind to cdk5. D shows cdk5 pulled down significantly less p35 in the presence of GFP-httEx1-74Q, compared with cdk5 in the presence of GFP-httEx1-23Q. However, comparable amounts of p35 and cdk5 were seen in the total lysates, and similar levels of cdk5 were immunoprecipitated in cells expressing 23Q or 74Q ( D, lanes 1 and 3). To confirm that polyQ-expanded htt impeded the p35–cdk5 interaction, we cotransfected p35–cdk5 and increasing amounts of httEx1-74Q into COS-7 cells, and then anti-cdk5 was used to pull down p35 and httEx1-74Q in cell lysates ( E). The levels of p35 that were pulled down by cdk5 decreased with the increasing expression of httEx1-74Q.
Next, we tested if endogenous htt phosphorylation was affected in our HD mice that express the first 171 residues of mutant htt because cdk5 activity is reduced in these mice ( A). Htt S434 phosphorylation (phospho-htt/total htt ratio) was significantly reduced in 8- (presymptomatic) or 17-wk-old (symptomatic) HD mouse brain lysates, compared with nontransgenic age-matched littermates ( F; by 33% and 17%, respectively). The apparent differences in the reduction in htt phosphorylation at 8 and 17 wk may simply be because the experiments at these time points were performed at different times and on different blots.
Htt588 or full-length htt stabilizes p35–cdk5 interaction and expanded polyQ of mutant htt decreases this stabilization
Because htt toxicity is believed to be exposed after cleavage, we tested if mutant htt588 mutant full-length htt reduced the p35–cdk5 interaction. Surprisingly, we found that both httwt588 and httmu588 ( A) and full-length wthtt and muhtt ( B) increased the p35–cdk5 interaction. However, mu588 and mutant full-length were less effective than their wt counterparts (). However, exon1-23Q does not significantly alter the affinity of p35–cdk5 interaction, whereas exon1-74Q interferes with the interaction, which is consistent with (C–E). The cleavage of full-length htt and htt588 may explain why the mutant forms of these proteins are less effective than their wild-type counterparts at stabilizing the cdk5–p35 interaction because mutant htt exon 1 impairs p35–cdk5 binding. In addition, we consistently observed that more mutant htt than wild-type protein was pulled down using anti-cdk5 (J3) (), which we did not observe using anti-Flag antibody. The stronger binding is not nonspecific because neither httw588 nor httmu588 were pulled down by anti-cdk5 in httwt588- or httmu588-only transfected cell lysates. (Please note that it is almost impossible to achieve equal transfection efficiencies of exon 1 htt, htt588, and full-length htt in the same experiment due to the vastly different sizes of the expression vectors.)
Figure 8. Htt588 or full-length htt stabilizes p35–cdk5 interaction, and expanded polyQ of mutant htt compromises this stablization. (A) p35–cdk5 (0.75 μg each) were cotransfected into HeLa cells with 1 μg of empty vector (lane 1), (more ...)
The data in cell and mouse models suggest that the interaction between cdk5 and its activator p35 is impaired by NH2-terminal mutant htt, which accounts for the reduced cdk5 activity we observed in HD mouse brains. Thus, the ability of cdk5 to prevent htt cleavage by caspases may be partially abrogated in HD mouse brains.