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First published online 24 February 2009
doi: 10.1242/jcs.050013

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Huntingtin promotes cell survival by preventing Pak2 cleavage

Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK

View larger version (49K): [in this window] [in a new window]   Fig. 1. Pak2 is a Htt-binding protein. (A) Left panels: Myc-Pak2–vector (Myc-Pak2 along with empty vector), Myc-Pak2–FLAG-Htt316-488 (Myc-Pak2 along with a vector expressing residues 316-488 of wild-type Htt), Myc-Pak2–FLAG-Htt316-513 (residues 316-513 of wild-type Htt), Myc-Pak2–FLAG-Htt316-552 (residues 316-552 of wild-type Htt) or Myc-Pak2–FLAG-Htt552 (first 552 residues of Htt) were co-transfected into HeLa Tet-on cells. Doxycycline (1 µg/ml) was added to the media to induce Myc-Pak2 expression following transfection. After 24 hours, cells were lysed and anti-FLAG antibody was used for immunoprecipitation. Anti-Myc was used to detect Pak2 (top) and anti-FLAG was used to detect Htt variant proteins (bottom). Right panels: Myc-Pak2–vector, Myc-Pak2–FLAG-Htt190 (residues 1-190 of wild-type Htt), Myc-Pak2–FLAG-Htt315, Myc-Pak2–FLAG-Htt552 or Myc-Pak2–FLAG-Htt588 were co-transfected into HeLa Tet-on cells. After 24 hours, cells were lysed and anti-FLAG antibody was used for immunoprecipitation. Anti-Myc was used to detect Pak2 (top) and anti-FLAG was used to detect Htt variant proteins (bottom). (B) Schematic showing the domain of Htt that binds Pak2: –, nonbinding; +, binding; ++, stronger binding. (C) Mouse brain lysate was immunoprecipitated with anti-FLAG antibody as a control (NS; lane 2) or with anti-Htt antibody (MAB2166; lane 3), and the blot probed with anti-Pak2 antibody (left column) and anti-Htt antibody (middle and right columns). Lane 1 was loaded with 8% of total lysate. (D) Mouse brain lysate was immunoprecipitated with anti-GST antibody as a control (NS; lane 2) or anti-Pak2 antibody (lane 3) and the blot probed with anti-Htt antibody (left column) and anti-Pak2 antibody (right column). Lane 1 was loaded with 6% of total lysate. FL-htt, full-length Htt.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Wild-type Htt prevents Pak2 cleavage in cells. (A) Myc-Pak2 was transfected along with increasing amounts of Htt588 into HeLa Tet-on cells. Total DNA in each transfection sample was kept constant. Transfected cells were treated with TNF+CHX for 4 hours. The first lane is the sample of cells transfected with Myc-Pak2 without treatment (un). The lysates were subjected to SDS-PAGE and blotting with anti-Myc antibody (top) to detect full-length Pak2 and N-terminally truncated Pak2 [tPak2 (N–)], and with anti-FLAG antibody (bottom). Both upper bands and lower bands were quantified. Blot is representative of data from three independent experiments. The ratios of cleaved to intact Pak2 are indicated here and in subsequent figures. (B) Myc-Pak2–empty vector and Myc-Pak2–Htt552 were transfected into HeLa Tet-on cells. Where indicated, cells were treated with epoxomycin (lanes 3, 4), and lactacystin (lanes 5, 6). Cell lysates were subjected to SDS-PAGE. Two different anti-Myc antibodies were used for probing the filter. The top panel shows monoclonal anti-Myc, 9E10; the bottom panel shows polyclonal anti-Myc. The bands and lanes on the top panel correspond to those on the bottom panel. Note that cells were not treated with death stimuli (TNF+CHX) in this experiment. (C) Pak2 was co-transfected into HeLa cells with either empty vector, Htt120, Htt190, Htt315, Htt552 or Htt588. After 18 hours, cells were treated with TNF+CHX for 4 hours. Full-length Pak2 and C-terminal Pak2 [tPak2 (C–)] were detected with anti-Pak2 (C-terminal) antibody by western blot. The blot is representative of data from three independent experiments (top panel). Bottom panel: the blot was detected with anti-FLAG, showing comparable expression levels of different Htt variants. Note that Htt588 and Htt552 exhibit three bands and two bands, respectively, due to caspase cleavage. (D) Myc-Pak2 was transfected into HeLa Tet-on cells. The four transfections were treated with DMSO (control), 20 µM z-VAD-fmk, 50 µM caspase-3 inhibitor-I (C3-I) or 50 µM caspase-8 inhibitor-I (C8-I) following transfection. After 20 hours, all transfected cells were treated with TNF+CHX for 5 hours. Cells were harvested and subjected to western blot with anti-Pak2 antibody. (E) Pak2 was co-transfected with empty vector or Htt552 into MCF-7 cells. After 20 hours, cells were treated with CHX (lanes 1, 2) or TNF+CHX (lanes 3, 4) for 6 hours. Pak2 was detected with anti-Pak2 antibody (C-terminal) in a western blot assay. Note that CHX on its own is not significantly pro-apoptotic under these conditions, as TNF needs to be present to cause cell death. The molecular mass of the Pak2 C-terminal fragment was 34 kDa. The blot is representative of data from three independent experiments. (F) Pak2 was transfected into MCF-7 cells. After 20 hours, cells were treated without (lanes 1, 2) or with (lanes 3, 4) TNF+CHX for 6 hours. At 3 hours after transfection, when transfection media was replaced with complete media, C8-I (50 µM) was added to cells (lanes 2, 4). Pak2 was detected with anti-Pak2 antibody (C-terminal) in a western blot assay.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Pak2 cleavage, caspase-3 activation and cell death in Htt+/+ or Htt–/– ES cells under serum-withdrawal conditions. (A) Htt+/+ and Htt–/– mouse ES cells were cultured in media containing serum and LIF (lanes 1, 2). For assay of Pak2 cleavage, cells were starved in media containing neither serum nor LIF for 24 hours (lanes 3, 4). Cells were harvested and subjected to SDS-PAGE and blotting with anti-Pak2 antibody (C-terminal). The blot is representative of data from three independent experiments. (B,C) Htt+/+ or Htt–/– ES cells were each divided into two sets and cultured in complete ES cell medium for 2 days. One set of Htt+/+ or Htt–/– ES cells were starved in serum-free DMEM for 18 hours (lanes 3, 4). The other set of ES cells was continuously cultured in complete ES cell medium (lanes 1, 2). (B) The cell lysates were subjected to SDS-PAGE and western blotting with anti-caspase-3 antibody. FL, full length. (C) Cell death was evaluated by staining with Trypan blue. Samples are in triplicate. Comparisons were performed using the Student's t-test; error bars are s.d.; P1=0.0602, *P2=0.0265, *P3=0.0039.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Wild-type Htt interacts more strongly with Pak2 than does muHtt. (A) Myc-Pak2–vector, Myc-Pak2–FLAG-Htt588 (wild type, 17 glutamine repeats) or Myc-Pak2–FLAG-muHtt588 (138 glutamine repeats) were transfected into HeLa Tet-on cells. After 20 hours, the transfected cells were harvested and lysed. The cellular lysates were subjected to anti-FLAG (M2) agarose immunoprecipitation. The immunoprecipitates (lanes 4-6) and total lysates (lanes 1-3) were used for SDS-PAGE. The blot was probed with anti-Myc (top panel) and anti-FLAG (bottom panel) antibodies. (B) Quantification of the interaction strength between Pak2 and either wild-type Htt588 or muHtt588 was made using data from Fig. 2A and two other similar experiments: y-axis shows ratio of immunoprecipitated (IP) Pak2 to total Pak2. The ratio of IP Pak2 to total Pak2 when wild-type Htt588 was used for Pak2 immunoprecipitation was set as 1. Data were from three independent experiments. Comparison was performed using the Student's t-test; error bar is s.d.; *P=0.00855.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Mutant Htt reduces Pak2 cleavage in cells. (A) Pak2 was transfected with empty vector (lanes 1-3), wild-type (wt) Htt588 (lanes 4-6) or muHtt588 (lanes 7-9) into HeLa cells. Transfected cells were treated with TNF+CHX for 0, 3 or 6 hours. The lysates were subjected to SDS-PAGE and blotting with anti-Pak2 antibody (C-terminal) (top panel) and anti-FLAG antibody for Htt (bottom panel). The molecular mass of the Pak2 C-terminal fragment was 34 kDa. Blot is representative of data from four independent experiments. [We believe that 50 kDa is likely to be a nonspecific band as it was only present with certain batches of the antibody under certain conditions (incubation time and washing).] (B) Myc-Pak2 was transfected with empty vector (lanes 1-3), wild-type Htt588 (lanes 4-6) or muHtt588 (lanes 7-9) into HeLa Tet-on cells. Transfected cells were treated with TNF+CHX for 0, 3 or 6 hours. The lysates were subjected to SDS-PAGE and blotting with anti-Myc antibody to detect an N-terminal Pak2 fragment (top panel), and with anti-FLAG antibody for Htt (bottom panel).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Htt prevents Pak2 cleavage by caspase-3 in vitro. (A) Myc-Pak2–vector or Myc-Pak2–Htt552 was transfected into HeLa Tet-on cells. Cells were harvested and lysed. Cell lysates were cleaved by caspase-3 in vitro (37°C, 1 hour) (lanes 3, 4) or mock-cleaved in vitro (buffer, 37°C, 1 hour) (lanes 1, 2). The cleaved lysates were subjected to western blot and probed with an anti-Myc antibody. The molecular mass of the Pak2 N-terminal fragment was 29 kDa; * indicates a nonspecific band. (B) The blots for experiments performed as in A were quantified with a ChemiImager. The ratio of caspase-3-cleaved Pak2 to full-length (FL) Pak2 in the Pak2-alone lysate was set to 1. The relative level of caspase-3-cleaved Pak2 to full-length Pak2 in Pak2-Htt552 cells is shown. Five independent experiments were performed. Comparison was performed using the Student's t-test; error bar is s.d.; ***P=0.00004. (C) Assays of caspase-3 activity were performed for the samples in A; n=3. (D) Pak2-vector or Pak2-Htt552 was transfected into HeLa cells. Cells were harvested and lysed. Cell lysates were cleaved by caspase-3 in vitro (37°C, 1 hour) (lanes 3, 4) or mock-cleaved in vitro (buffer, 37°C, 1 hour) (lanes 1, 2). The cleaved lysates were subjected to western blot and detected with anti-Pak2 antibody that recognizes the C-terminus of Pak2. (E) The blots for the experiments performed as in D were quantified with a ChemiImager. The ratio of caspase-3-cleaved Pak2 to full-length Pak2 in the Pak2-alone lysate was set to 1. The relative value of caspase-3-cleaved Pak2 to full-length Pak2 in Pak2-Htt552 cells is shown. Three independent experiments were performed. Comparison was performed using the Student's t-test; error bar is s.d.; *P=0.00483. (F) Assays for caspase-3 activity were performed for the samples in D; n=3.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Htt prevents caspase-8-mediated Pak2 cleavage in vitro. (A) Pak2-vector or Pak2-Htt552 was transfected into MCF-7 cells. Cells were harvested and lysed. Cell lysates were cleaved by caspase-8 in vitro (37°C, 1 hour) (lanes 3, 4) or mock-cleaved in vitro (buffer, 37°C, 1 hour) (lanes 1, 2). The cleaved lysates were subjected to western blot and detected with anti-Pak2 antibody (C-terminal). (B) Quantification of data from Fig. 6A along with additional replicates. The ratio of caspase-8-cleaved Pak2 to full-length Pak2 in Pak2-alone lysate is set as 1. The relative value of caspase-8-cleaved Pak2 to full-length Pak2 in Pak2-Htt552 cells is shown. Three independent experiments were performed. Comparison was performed using the Student's t-test; error bar is s.d.; *P=0.00505.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Htt protection of Pak2 cleavage by caspases is important for the anti-apoptotic role of Htt. (A) GFP-Pak2–vector, GFP-Pak2–Htt588, GFP-Pak2–Htt552, GFP-Pak2–Htt315 or GFP-Pak2–Htt190 was transfected into HeLa Tet-on cells. After 24 hours, cells were treated with TNF+CHX for 4 hours. Cells were then fixed and cell death evaluated in GFP-positive cells. Note that the different Htt constructs express at similar levels (Fig. 3C and supplementary material Fig. S2). Comparisons were performed using the Student's t-test; error bars are s.d.; NS, not significant, *P1=0.0237, P2=0.0192. (B) GFP-Pak2p34–vector or GFP-Pak2p34–Htt588 was transfected into HeLa Tet-on cells. After 48 hours, cells were fixed and cell death evaluated in GFP-positive cells. Comparisons were performed using the Student's t-test; error bars are s.d. (C) Transfection reagent was used for mock transfection. Control siRNA or Pak2 Smartpool (SP) siRNA was transfected into HeLa cells at 50 nM. After 48 hours, cells were harvested and blots were probed with anti-Pak2 (top panel) and anti-tubulin (bottom panel) antibody. (D) HeLa cells were treated with control siRNA or Pak2 siRNA for 48 hours and then transfected with Htt588. After a further 24 hours, cells were treated with TNF+CHX for 4 hours to induce cell death. Cells were fixed and stained with anti-FLAG antibody to detect Htt overexpression. Cell death was assessed in Htt588-transfected cells. Comparisons were performed using the Student's t-test; error bars are s.d.; NS, not significant, *P1=0.00884, *P2=0.0074, *P3=0.00712.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Htt protection against TNF-induced cell death is dependent on Pak2 cleavage. (A) Control siRNA, or human Pak2 siRNA-1, siRNA-2, siRNA-3 or Smartpool (SP) (50 nM) was transfected into HeLa cells. After 48 hours, cell lysates were subjected to SDS-PAGE and western blot. The membrane was probed with anti-Pak2 (top panel) and anti-tubulin (bottom panel) antibodies. (B) Control siRNA, or human Pak2 siRNA-1, siRNA-2, siRNA-3 or Smartpool (SP) (50 nM) was transfected with mouse Pak2 (mPak2) (1 µg) into HeLa cells. As a control, EGFP was also co-transfected with the various siRNAs and mPak2. After 48 hours, cell lysates were subjected to SDS-PAGE and western blot. The membrane was probed with anti-Pak2 (top panel), anti-GFP (middle panel) and anti-tubulin (lower panel) antibodies. (C) Mouse wild-type Pak2 (lanes 1, 3, 5) or Pak2-D212E (lanes 2, 4, 6) was transfected into HeLa Tet-on cells. One group of Pak2-transfected or Pak2-D212E-transfected HeLa Tet-on cells were treated with TNF+CHX for 5 hours (centre column). Another group of Pak2-transfected or Pak2-D212E-transfected HeLa Tet-on cells were treated with the potent cell-death agent Staurosporine (STS) (1 µM) for 6 hours (right column). A third group of Pak2-transfected or Pak2-D212E-transfected HeLa Tet-on cells were the untreated controls (left column). Cell lysates were subjected to SDS-PAGE, and western blot was probed with anti-Pak2 antibody (C-terminal). Note that the top bands are the uncleaved Pak2, whereas the lower bands are the cleavage products. The positions of the lower bands differ slightly, as samples were not all from the same gels. (As mPak2 expression is comparatively high under these conditions, we loaded comparatively low amounts of total protein on the gels and used short exposures on these blots, which made the endogenous products below the limits of detection.) (D) pEGFP-C1 was co-transfected into HeLa cells with the indicated plasmids (the ratio of Pak2 or Pak2-D212E to Htt588 was 1:1.5 and to vector-EGFP was 1:0.3). Human Pak2 siRNA-3 (50 nM) was also co-transfected with these plasmids. After 48 hours, the cell death of GFP-positive cells was evaluated under a fluorescent microscope. Data are from triplicates. Comparisons were performed using the Student's t-test; error bars are s.d.; *P=0.0157.

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