(A) VICTR37 retroviral gene trap inserted into intron 1 of the murine PACS-2 gene. The gene trap (PACS-2^{GT(OST426086)}) produces a truncated protein chimera containing only the first 40 amino acids of the 889 amino acid PACS-2. Arrows above exons 1 and 11 depict PCR primers used in panel B. SD, splice donor site; SA, splice acceptor. (B) RNA was isolated from PACS-2 WT (PACS-2^+/+), heterozygote (PACS-2^{+/GT(OST426086)}, hereafter called PACS-2^+/-), or PACS-2 gene trap ((PACS-2 ^{GT(OST426086)/GT(OST426086)}, hereafter called PACS-2^-/-) littermates and analyzed by RT-PCR to detect PACS-1, PACS-2 and ATP citrate lyase (ACYL, positive control). (C) Tissues from WT, +/- or -/- mice were analyzed by western blot for PACS-2. (D) WT and PACS-2^-/- mice (n = 4 in each group) were injected with AdGFP and mouse AdTrail. TUNEL and c-caspase-3 positive cells were counted in liver sections from WT and PACS-2^-/- mice were visualized (magnification, 200x) and quantified as the number of TUNEL- and cleaved caspase 3-positive cells per 20X field (graphs). Liver steatosis was detected by Oil Red O staining. (E) WT and PACS-2^-/- mice (n = 4 in each group) were injected with the mAb Jo2 to stimulate Fas-mediated apoptosis or with LPS /GalN to stimulate TNFα-mediated apoptosis and TUNEL-positive nuclei were counted (original magnification 200x).

(A) Diagram of PACS-2 showing the N-terminal region (NTR), cargo-binding region (FBR), which contains Ser₄₃, the middle region (MR), which contains Ser₂₄₄, Ser₄₃₅ and Ser₄₃₇, and the C-terminal region (CTR). (B) The indicated GST-fusion proteins were incubated with [γ-³²P]-ATP and constitutively active HA-Akt (WT) or kinase-dead HA-Akt (KD). Con, immunoisolation from mock-infected cells. Left, autoradiography; Right, input. (C) The indicated GST-fusion proteins were incubated with [γ-³²P]-ATP and immunoisolated HA-Akt as described in panel B. Top, autoradiography; Bottom, input. (D) Peptides from in-gel limited trypsin digestion of HA-tagged PACS-2 were evaluated for candidate phosphopeptide by mass spectrometry. A phosphopeptide at [M+2H]=450.7, corresponding to the PACS-2 tryptic peptide RSTpS₄₃₇LKER with one phosphorylation, was identified by nanoLC-MS/MS. (E) Left; Brain cytosol from WT and PACS-2^-/- mice was incubated with affinity purified anti-PACS-2 (Ab 832) and immunoprecipitated proteins were analyzed by western blot using anti-PACS-2 (Ab 193) or mAb 81 to detect pSer₄₃₇. Right; Liver and small intestine cytosol from WT mice were analyzed as described above. (F) Lysates from WT and Akt1^-/- MEFs were analyzed as described in panel E to detect pSer₄₃₇. (G) HCT116 cells were harvested in proliferative phase (untreated), serum-starved for 16 hr (starved), starved and refed with 20% serum for 3 hr (control), or starved and pre-treated with 1 μM PI-103, 20 nM RAD011, 1 μM BKM-120 or 20 nM BEZ-235 for one hr prior to refeeding with 20% serum for 3 hr. Cells were analyzed by western blot to detect total PACS-2 and PACS-2 pSer₄₃₇. The pSer₄₃₇:total PACS-2 ratio was calculated and normalized to the untreated sample. Data are represented as mean +/- SD.

(A) HCT116 cells expressing HA-tagged PACS-2 and myc-tagged 14-3-3ζ were treated with 25 ng/ml TRAIL for the indicated times. PACS-2ha immunoprecipitates were immunoblotted to detect co-precipitating 14-3-3ζ and pSer₄₃₇. (B) TAg-PACS-2^-/- cells were co-transfected with an eYFP-tagged caspase-3 reporter together with pcDNA3.1 (vector) or plasmids expressing HA-tagged PACS-2 or PACS-2S₄₃₇A alone or with 14-3-3ζ-myc. Top: Cells treated with 10 ng/ml mouse TRAIL for 16 hr were fixed and the number of nuclear eYFP-positive cells indicating activated caspase-3 were analyzed. Data are represented as mean +/- SEM. Bottom: Representative immunofluorescence images of the quantified data. Insets, co-expression of 14-3-3ζ-myc and HA-tagged PACS-2 or PACS-2S₄₃₇A. Scale bar, 20 μm. (C) TAg-PACS-2^-/- cells described in B were lysed, PACS-2 immunoprecipitated and co-precipitating 14-3-3ζ-myc detected by western blot.

(A) HCT116 cells were co-nucleofected with pmaxGFP and either a control siRNA (scr.) or PACS-2 siRNAs (#1, Qiagen, #2, Dharmacon) and then treated with 0, 10 or 100 ng/ml TRAIL for 16 hr. GFP⁺ cells were analyzed for apoptosis by flow cytometry. Data are represented as mean +/- SEM. Insets: Western blot of PACS-2 in control and PACS-2 knockdown cells. (B) Matched colorectal tumor (T) and adjacent normal (N) colonic tissue from eight volunteers with metastatic (samples 1-3) or stage 3 (samples 4,6 8-10) cancer were homogenized and 100 μg of total protein from each sample was analyzed by western blot. Arrow, PACS-2; *, 150 kDa PACS-2 immunoreactive protein of unknown identity. (C) A paraffin block of colonic tumor was sectioned and analyzed for PACS-2 expression by immunohistochemistry. Left, Normal colonic epithelium (N) and adjacent tumor (T). Right, Magnification of normal and tumor regions.

(A) Primary WT and PACS-2^-/- MEFs, as well as TAg-WT, TAg-PACS-2^-/-, TAg-PACS-2^V or TAg-PACS-2^R MEFs were treated with increasing concentrations of TRAIL for 16 hr and then analyzed for apoptosis by Annexin V/ propidium iodide staining. Inset, western blot of PACS-2 and DR5 expression in the cultures. Data are represented as mean +/- SEM. (B) Lysates from TAg-WT and TAg-PACS-2^-/- MEFs treated or not with 25 ng/ml TRAIL for 4 hr were incubated with the caspase-8 substrate Ac-IETD-AFC or the caspase-3 substrate Ac-DEVD-AFC and free AFC fluorescence was quantified. Data are represented as mean +/- SEM. (C) TAg-WT and TAg-PACS-2^-/- MEFs were treated with 50 ng/ml TRAIL for the times indicated and the cleavage of each apoptotic factor was determined by western blot. Actin, loading control. (D) TAg-WT and TAg-PACS-2^-/- MEFs were treated or not with 25 ng/ml TRAIL for 4 hr. Cells were permeabilized with digitonin and the cytosol supernatant (S) and membrane pellets (P) were separated by centrifugation. GRP78, organelle pellet marker; tubulin, cytosol marker. (E) Primary or TAg MEFs were treated with 100 ng/ml TNFα plus 1 μg/ml Actinomycin D or 250ng/ml FasL for 4 hr and analyzed as described in panel C. (F) TAg-WT and TAg-PACS-2^-/- MEFs were pretreated or not with vehicle (DMSO) or 1 μM PI-103 for 1 hr then treated with 50 ng/ml TRAIL for the indicated times and analyzed as described in panel C.

(A) PACS-2 was immunoprecipitated from rat brain cytosol and co-precipitating 14-3-3 was detected by western blot. (B) The indicated GST fusion proteins were incubated with ATP and constitutively active HA-Akt (WT) or kinase-dead HA-Akt (KD), then incubated with 14-3-3σ-his₆. Protein complexes were captured with glutathione-sepharose and bound 14-3-3σ-his₆ was detected by western blot. (C) (TMR)–labeled 16mer peptides containing pSer₂₅₉-Raf, PACS-2 Ser₂₄₄-PACS-2, pSer₂₄₄-PACS-2, Ser₄₃₇-PACS-2 or pSer₄₃₇-PACS-2 (1 nM) were incubated in triplicate with increasing concentrations of GST-14-3-3γ. Fluorescence polarization signals were recorded and used to calculate K_ds. (D) 14-3-3 isoforms were incubated with (TMR)-labeled pSer₄₃₇-PACS-2 or pSer₂₅₉-Raf peptides and analyzed as described in panel C. Calculated K_d values are shown.

(A) HeLa cells were nucleofected (Amaxa) with myc-TRPP2 together with either empty vector or HA-tagged PACS-2 or PACS-2S₄₃₇A. After 36 hr, the cells were fixed and processed for confocal microscopy using anti-myc (mAb 9E10, green) and anti-Golgin-97 (red) and visualized using Alexa-conjugated secondary antibodies. Right: Morphometric analysis. Error bars represent mean +/- SEM. Scale bar, 20 μm. (B) HeLa cells expressing myc-TRPP2 were treated with 20 ng/ml TRAIL for 4 hr and processed for confocal microscopy. Mitotracker (pseudocolored blue), anti-myc (green) and anti-Golgin-97 (red) are shown. *, apoptotic cell showing puncate myc-TRPP2 and collapsed mitochondria. Arrows, overlap of myc-TRPP2 and Golgin-97. Scale bar, 20 μm. (C) MCF-7:Bid-GFP cells were nucleofected (Amaxa) with pcDNA3.1 (vector) or plasmids expressing HA-tagged PACS-2 or PACS-2S₄₃₇A alone or with 14-3-3ζ-myc. The cells were then treated with vehicle or 20 ng/ml TRAIL for 4 hr. Left: The percent of cells containing Bid-GFP translocated to mitochondria (mitotracker, red) in random fields (300 cells minimum) were quantified. Data are represented as mean +/- SD. Right: Immunofluorescence of MCF-7:Bid-GFP expressing the indicated proteins and treated or not with TRAIL. Insets, co-expression of 14-3-3ζ-myc with HA-tagged PACS-2 or PACS-2S₄₃₇A. Scale bar, 20 μm.