PLSCR1 reverses onzin's ability to suppress p53 induction and G₂/M arrest. (A) The lysates in Fig. 9B were used to detect total p53 protein. As a control for protein loading, tubulin was also detected in each sample. (B) At the time of cell harvest shown in panel A, a separate aliquot of cells was stained with propidium iodide and subjected to flow cytometry to quantify the fraction of each cell population in G₀/G₁, S, and G₂/M. The numbers indicate the averages of triplicate determinations, and each figure displays a typical determination for that sample.

Region between amino acids 160 and 250 of PLSCR1 is required for its interaction with and inhibition of onzin. (A) Individual Rat1a-onzin clones (lanes 1 to 4) or control Rat1a-puro clones (lanes 5 to 8) expressing full-length PLSCR1, PLSCR1(160-250), or PLSCR1(1-163) were subjected to immunoblotting. Both onzin and PLSCR1 proteins were Myc epitope tagged and thus were detected simultaneously with the 9E10 anti-Myc MAb. (B) Coimmunoprecipitation of onzin and full-length PLSCR1 and PLSCR1(160-250). The Rat1a cell lines shown in panel A were lysed, immunoprecipitated with anti-PLSCR1 MAb and immunoblotted using the 9E10 anti-Myc MAb. + and − indicate the expression or lack thereof of the indicated proteins based on the immunoblot analysis depicted in panel A. Note that onzin coimmunoprecipitated with full-length PLSCR1 and PLSCR1(160-250) but not with PLSCR1(1-163).

Coimmunoprecipitation of onzin and PLSCR1 from Cos7 cells. (A) Myc epitope-tagged full-length murine onzin or the del28-38 mutant (Fig. 1) was coexpressed in Cos7 cells along with full-length human Flag epitope-tagged PLSCR1. Following lysis, each protein was detected by immunoblotting using antibodies specific for the indicated epitope tags. Blots were also probed with an antiactin monoclonal antibody as a control for protein loading. (B) The lysates from panel A were immunoprecipitated with the anti-Flag antibody or a control IgG. Precipitates were then immunoblotted and onzin protein was detected with the anti-Myc tag MAb. Note that the del28-38 onzin mutant did not interact with PLSCR1. (C) Myc epitope-tagged, onzin-overexpressing NIH 3T3 fibroblasts were generated by retroviral infection. Total lysates were then immunoprecipitated with a Myc epitope antibody or control IgG and blotted for associated endogenous PLSCR1. (D) The lysates in panel C were first precipitated with the anti-PLSCR1 MAb or control IgG. The blot was then probed with the anti-c-Myc MAb. Preliminary studies indicated that the cells expressed equivalent levels of wild-type and onzin(Δ28-38) protein (not shown).

Expression and colocalization of onzin and PLSCR1 in mammalian cells. (A) Full-length murine PLSCR1 was expressed as an N-terminal fusion with the enhanced yellow fluorescent protein analog of GFP (EYFP) and full-length murine onzin was similarly fused to the enhanced blue variant (EBFP). Each fusion protein was then expressed individually in Cos7 cells, which were fixed 16 h later and viewed by fluorescence microscopy. In columns 2 and 3, cells were deprived of serum or exposed to 125 nM etoposide for 12 h prior to fixation. Typical images are shown. In other experiments, properly sized proteins representing the fusions were observed in the same cells by immunoblotting using an MAb against GFP (not shown). (B) The two proteins were coexpressed, and their individual subcellular localizations were determined by imaging each one singly (28). The bottom row shows the merged images. All of the images depicted here are representative of the appearance of >200 transfected cells.

Domain from 160 to 250 of PLSCR1 is sufficient to inhibit onzin's biological effects. (A) Each of the clones described in the legend to Fig. 7 was grown to approximately 50% confluence and deprived of serum, and the residual viable cells were quantified on a daily basis. (B) Cells were grown as described for panel A except that fresh medium containing 125 nM etoposide (VP16) was added on day 0. Residual viable cells were quantified on subsequent days. (C) Cells were seeded at equivalent low densities (2 × 10³ cells/well) in medium containing 1% fetal calf serum. Subsequent cell counts were performed at the indicated times. In panels A to C, all points represent the average of triplicate determinations ± 1 standard error. (D) Each of the indicated clones was seeded as described in the legend to Fig. 5D and subsequently incubated in medium containing 1 μM 6-thioguanine. Methylene blue-stained colonies were then visualized on days 5 and 6.

Yeast two-hybrid analysis identifies the onzin-interacting domain of human PLSCR1. (A) Previously identified functional domains of human PLSCR1 are shown on the depiction of the full-length, 318-amino-acid molecule. These domains include two PPXY-containing WW-binding domains, a PXXP-containing SH3-binding domain, phosphotyrosines serving as putative SH2- and PTB-binding motifs, the CCCPCC-containing Cys palmitoylation site, the atypical nuclear localization signal (GKISKHWTGI), the Ca²⁺ binding site, and the predicted type 2b transmembrane (TM) helix (4, 36). (B) Each of the indicated PLSCR1 mutants plus the pGAD vector containing full-length PLSCR1 and the empty pGAD-T7 parental vector was expressed in the AH109-pGBKT7-onzin yeast strain. In the 5C→5A PLSCR1 mutant, the palmitoylation signal CCCPCC was altered to AAAPAA (36). The 5C→5A/K→A mutation contains the palmitoylation site mutation plus mutation K258→A within the nuclear localization signal, which abrogates nuclear trafficking of the protein (4). The table shows the results of yeast growth assays on quadruple-knockout plates and of quantitative β-galactosidase assays as described in the legend to Fig. 1. (C) Expression of human activation domain-PLSCR1 fusion proteins. Western blotting was performed as described for Fig. 1 but were probed with an MAb against the HA tag, located between the activation domain and PLSCR1 moieties. Arrowheads indicate the positions of the fusions proteins.

PLSCR1 inhibits onzin's ability to interact with and regulate Akt1 and Mdm2. (A) Coimmunoprecipitation experiments demonstrating the association between onzin and Akt1 and Mdm2 and its reversal by PLSCR1. The indicated cell lines were lysed and immunoprecipitated with antibodies to either Akt1 or Mdm2 (+) or with a control IgG (−) as previously described (28). Immunoblotting was then performed on the precipitates with an anti-Myc monoclonal antibody to detect Myc epitope-tagged onzin. (B) PLSCR1 overexpression blocks onzin's ability to promote phosphorylation-dependent activation of Akt1 and Mdm2. Each of the indicated cell lines was exposed to 125 nM etoposide for the indicated times. After lysis, 50 μg of total lysate from each time point was then subjected to immunoblotting for the indicated proteins or phosphoproteins. In other experiments, blots were stripped and reprobed with an antitubulin MAb to confirm the loading of equivalent amounts of total lysate (see Fig. 10). In the case of pAkt1, day 0 and day 2 ratios of proteins were compared by densitometric scanning and were typical of several independent experiments. These ratios ranged from 1.2 to 2.3 for rows 1, 3, 4, 5, 6, and 7 and were 0.7 and 0.5 for rows 2 and 8, respectively.

shRNA-mediated knockdown of endogenous PLSCR1 in 32D myeloid cells enhances their proliferation and apoptotic resistance. (A) Endogenous PLSCR1 levels in clonal populations of 32D cells isolated following stable transfection with a control vector or with an shRNA vector targeting PLSCR1. Immunoblotting was performed using MAbs directed against PLSCR1 or tubulin as a control for protein loading. (B) Growth curves of 32D clones. Each of the indicated clones was seeded at a concentration of 2 × 10³ cells/ml in standard RPMI growth medium supplemented with 10% fetal bovine serum and IL-3. On each subsequent day, the total number of cells/dish was counted in triplicate plates. Each point represents the average of these determinations ± 1 standard error. Similar behaviors were seen with several additional single-cell-derived clones (not shown). (C) Logarithmically growing 32D cells (>90% viability) were washed twice and replated in IL-3-deficient medium at a concentration of 5 × 10⁵ cells/ml. Viability was then determined 20 h later on a Vi-Cell apparatus. A minimum of 2,000 cells were counted for each determination. The results shown represent the average percentage of viable cells from triplicate plates ± 1 standard error. (D) Cells were plated as described for panel C except that the medium contained IL-3 plus 125 nM adriamycin (ADR). Cell viability was determined as described for panel C.

Identification of the PLSCR1-interacting domain of onzin. (A) The cartoon shows the deletion mutants of murine onzin that were expressed as Gal4 DNA-binding domain fusion proteins in the pGBK-T7 vector. The critical PLSCR1-interacting domain residing between amino acids 28 and 38 is indicated by the black box, with the actual sequence displayed above the depiction of the full-length molecule. The table summarizes the ability of yeast cells expressing each of these mutants and coexpressing either a Gal4 activation domain-PLSCR1 fusion or the activation domain alone (e.g., the empty pGADT7 vector) to grow on medium lacking histidine and adenine and to express β-galactosidase. Each β-galactosidase assay was performed in triplicate on several occasions, and the results of a typical assay are shown, with the activity of the full-length onzin-PLSCR1 interaction normalized to 1. In all cases standard errors were <5% (not shown). To perform β-galactosidase assays on yeast cells expressing onzin mutants which did not confer Ade⁺ His⁺ prototrophy, these nutrients were included in the growth medium. (B) Western blots showing the expression of the indicated Gal4 DNA-binding domain-onzin fusion molecules. We analyzed 50 μg of protein from total yeast lysates in each case (21). Fusion proteins were detected with an MAb directed against a short Myc epitope residing between the binding domain and onzin moieties. The positions of the fusion proteins are indicated by arrowheads. Note that despite a somewhat lower level of fusion protein in lane 3, this yeast strain expressed the highest levels of β-galactosidase.

PLSCR1 negates the proliferative and antiapoptotic phenotype conferred by onzin. (A) Rat1a cells were stably transfected with plasmids encoding Myc-tagged PLSCR1 and/or Myc-tagged onzin. Individual clones were then picked, expanded, and examined for the expression of each protein by immunoblotting (25 μg of protein/lane). Control blots for actin confirmed the presence of equivalent amounts of protein in each lane. (B) Equal numbers of cells from the indicated clones were seeded and then grown until approximately 50% confluence. They were then deprived of serum (top panel) or exposed continuously to 125 nM etoposide (bottom panel). Viable cells were then enumerated on successive days. Each point represents the average of triplicate determinations ± 1 standard error. (C) We plated 2 × 10³ cells from the clones depicted in panel B in standard growth medium containing 1% fetal bovine serum. Total cell counts were then performed in triplicate at the indicated times and are depicted here as an average ± 1 standard error. Several additional clones expressing PLSCR1 at levels comparable to those depicted in panel A behaved similarly to the clones depicted here and in panel B (not shown). (D) Each of the indicated cell lines was seeded at 10⁵ cells/plate and grown for 2 days, at which time the medium was changed and 6-thioguanine was added to a final concentration of 1 μM for 5 additional days. Plates were then stained with methylene blue as previously described (28). Very similar results were obtained with each of the clones shown in panel A (not shown).