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First published online 20 March 2007
doi: 10.1242/jcs.000018

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Mechanisms of the HRSL3 tumor suppressor function in ovarian carcinoma cells

Irina Nazarenko^*, Reinhold Schäfer and Christine Sers

Molecular Tumor Pathology, Institute of Pathology, University Medicine Charité Berlin, Schumannstrasse 20/21, 10117 Berlin, Germany

View larger version (37K): [in this window] [in a new window]   Fig. 1. The N-terminus of the HRSL3 protein (HREV1) is important for the binding of HRSL3 and PR65. (A) Left panel: proteins were immunoprecipitated with an anti-V5 antibody, and subsequently detected with anti-HA (top) and anti-V5 (bottom) antibodies. Lanes (from the left): 1, total cell lysate; 2, immunoprecipitation from cellular extracts harboring both proteins; 3, 4, immunoprecipitation from cellular extracts harboring PR65V5 or HREV1dCHA, respectively. Right panel: proteins were immunoprecipitated with an anti-HA antibody and subsequently detected with anti-V5 (top) antibody. The efficiency of the immunoprecipitation was controlled using an anti-HA antibody (bottom). Lanes (from the left): 1, immunoprecipitation from cellular extracts harboring both proteins; 2, 3, immunoprecipitation from cellular extracts harboring PR65V5 or HREV1dCHA, respectively. (B) To prove whether PR65 directly interacts with HRSL3, cleared COS-7 cell lysates were incubated with either GST or GST-HREV1dC proteins coupled to GST-Sepharose. Relative amounts of the proteins were analyzed using western blotting against PR65 and GST antibodies. (C) The HREV1FL expression construct carries wild-type full-length HRASL3 cDNA. Four domains of the HRSL3 protein are indicated: a proline-rich domain at the N terminus of the protein (PD), a HWAIY domain containing a conserved histidine (His23), an NCE domain containing a conserved cystein (Cys112), and a membrane binding domain (MBD). In the HREV1dCHA expression construct, 29 C-terminal amino acids are substituted by the HA-epitope. AWAIdCHA carries a mutation His23Ala; NSEdCHA carries a mutation Cys112Ser; dNdCHA harbors a deletion of the N-terminal proline-rich region. (D) COS-7 cells were cotransfected with the following expression plasmids: PR65V5 and HREV1dCHA, PR65V5 and HREV1-AWAIYdCHA, PR65V5 and HREV1-NSEdCHA, and PR65V5 and HREV1-dNdCHA. As negative controls, plasmids containing HA or V5 epitopes only were transfected (–). (Left panel) Top: proteins were immunoprecipitated with an anti-V5 antibody and subsequently analyzed with an anti-HA antibody. HREV1dCHA, HREV1-AWAIdCHA and HREV1-NSEdCHA associate with PR65V5, whereas the HREV1-dNdCHA deletion mutant is unable to interact with PR65V5. Bottom: incubation with an anti-V5 antibody demonstrates equal amounts of the immunoprecipitated PR65V5 protein. (Right panel) Top: proteins were immunoprecipitated with an anti-HA antibody and subsequently analyzed with an anti-V5 antibody. The PR65V5 protein was obtained in a complex with HREV1dCHA, HREV1-AWAIdCHA and HREV1-NSEdCHA. By contrast, no PR65V5 was detected in the immunoprecipitated HREV1-dNdC complex. Bottom: immunoprecipitation was controlled using an anti-HA antibody and demonstrates equal amounts of the HA-tagged HRSL3 proteins.

View larger version (29K): [in this window] [in a new window]   Fig. 2. HRSL3 (HREV1) interacts with the endogenous regulatory, but not the catalytic, subunits of PP2A. (A) Protein extracts from several cell lines were analyzed by immunoblotting against an HRSL3-specific antibody described previously as H-REV107-1. As a control, COS-7 cells transiently transfected with an HRASL3 expression plasmid, were used. PR36 and PR65 were detected with appropriate antibodies. Actin was used as a loading control. (B) The PR65 immunocomplex was recovered from OVCAR-3 cells transiently transfected with an HRASL3 expression plasmid (HREV1FL). Immunoprecipitated proteins (IP), and protein extracts used for the immunoprecipitation (Input) were subjected to SDS-PAGE and western blot analysis. Presence of the regulatory PR65 (top) and the catalytic PR36 (middle) subunits was demonstrated by immunoblotting with appropriate antibodies. Application of an anti-HRSL3 specific antibody (bottom) revealed the presence of the HRSL3 protein in the PR65 protein complex (second lane) but not in the control immunoprecipitation (third lane), confirming the interaction between HRSL3 and endogenous PR65. (C) OVCAR-3 cells were transiently transfected with HREV1FL, dNdC and pcDNA3 as a control. Forty-eight hours post-transfection immunoprecipitation was performed with a PR65-specific antibody (left panel) and with a PR36,-specific antibody (right panel). Presence of the HRSL3 full-length and truncated proteins, PR65,, PR36, and B56 in the protein extracts used for immunoprecipitation (Input) and in the precipitated protein complexes was analyzed using western blotting with the appropriate antibodies. *Decreased amount of the PR36 proteins in the PR65 protein complex in the presence of HRSL3 was semiquantitatively evaluated (see bar chart below) using ImageJ freeware. The ratio between the amounts of immunoprecipitated PR36 and the light chain of the PR65 antibody is shown on top of the bars. (D) OVCAR-3 cells were transiently transfected with HREV1FL-V5 and incubated with PR65, and V5-specific antibodies. Then the cells were stained with DAPI to visualize nuclei (blue), with Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies to visualize the distribution of the PR65 (red) and the HRSL3-V5 (green) proteins, respectively using confocal microscopy. (Merge) Yellow color indicates a colocalization of the HRSL3 and the PR65 proteins (overlay of red and green) in the cytoplasm. (E) OVCAR-3 cells were transiently transfected with HREV1FL and incubated with PR36, and HRSL3-specific antibodies. Then the cells were stained with DAPI to visualize nuclei (blue), with Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies to visualize distribution of the PR36 (green) and the HRSL3 (red) proteins, respectively using confocal microscopy. (Merge) Absence of yellow color indicates no colocalization of the HRSL3 and the PR36 protein in the cells.

View larger version (14K): [in this window] [in a new window]   Fig. 3. HRSL3 suppresses the PP2A catalytic activity. (A) OVCAR-3 cells were transiently transfected with HREV1FL and HREV1-dNdC, transfection with pcDNA3 was used as a negative control, and treatment with 10 nM okadaic acid as a positive control. Forty-two hours after transfection cells were lysed and immunoprecipitated with an anti-PR65 antibody. Phosphatase activity of the immunoprecipitates was measured as the release of free phosphate (pmol/minute; y axis). Six independent experiments were performed, and included into a statistical analysis. Significance of the obtained results was assessed using an F-test. The difference between HREV1FL and dNdC, and HREV1FL and pcDNA3 values was considered as significant (P=0.02). (B) Immunoprecipitates used for the measurement of the phosphatase activity in the presence of HRSL3 and its interaction-deficient mutant were controlled by western blotting using HRSL3- and PR65-specific antibodies.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Both HRSL3-dependent PP2A inhibition and siRNA-mediated suppression of PP2A regulatory subunit A, induce apoptosis via a caspase-dependent pathway. (A) Cleaved caspase-3 was detected by immunoblotting using appropriate antibodies. Total amount of caspase-3 was used as a loading control. In all experiments HRSL3 expression was controlled by immunoblotting with the HRSL3 specific antibody. Application of the caspase inhibitor zVAD-fmk leads to the abrogation of caspase cleavage. (B) OVCAR-3 cells were electroporated with the HREV1FL, dNdC expression plasmids. Twelve hours after transfection, cells were treated with 40 µM caspase inhibitor zVAD-fmk, if indicated. Alternatively, cells were treated with OA (10 nM), OA with zVAD-fmk (40 µM), or vehicle DMSO only. Seventy-two hours after transfection or treatment, nuclear DNA content was measured by flow cytometry. The relative number of cells displaying an apoptotic, sub-G1 content, is given between the marker bars. DNA histograms are representative of two independent experiments. (C) OVCAR-3 cells were transfected with PR65- and PR65-specific siRNAs individually, together, and treated with caspase inhibitor zVAD-fmk (40 µM), if indicated. The level of the PR65 protein and cleavage of caspase-3 were controlled 48 hours after transfection (upper panel). Efficiency of the PR65-specific RNA interference was verified using RT-PCR 12 and 24 hours after transfection. As a control, GAPDH mRNA was amplified (lower panel). (D) OVCAR-3 cells were transfected with PR65- and PR65-specific siRNAs individually or in combination. Apoptosis was analyzed at the single-cell level by measuring the nuclear DNA content. The relative number of cells displaying a sub-G1 DNA content is given between the marker bars. DNA histograms are representative of three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 5. HRSL3 does not influence the stability of PP2A. (A,B) OVCAR-3 cells were transfected with PR36-, PR36-, PR65- or PR65-specific siRNAs, or with the HRSL3-expressing plasmid (HREV1FL). Relative amounts of the proteins were controlled 24 and 48 hours after transfection using western blotting against PR65 or PR36 proteins.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Expression of the full-length HRSL3 protein (HREVFL) was controlled by incubation with the HRSL3-specific antibody (H-REV107-1). HRSL3 regulates PKC, which is required for HRSL3-induced apoptosis. (A) Phosphorylation of the indicated signaling proteins was analyzed by immunoblotting using antibodies specific for their phosphorylated forms. Immunoblotting against total amount of the proteins was used as a loading control. Expression of the HRSL3 protein, full-length and truncated form, was controlled by incubation with the HRSL3-specific antibody. (B) OVCAR-3 cells were either treated with 1, 10 and 20 nM of OA and with an inhibitor of the PI 3-kinase (LY294002; LY), or transfected with HRSL3. To determine the concentration of OA that inhibits PP2A activity only partially, phosphorylation of MEK1/2 and ERK1/2 kinases was examined using the corresponding antibodies (left panel). Then caspase-9 cleavage was analyzed (right panel). Actin- and caspase-9-specific antibodies were used as loading controls. (C) Phosphorylation of PKC is induced by treatment with both OA and HRSL3. Cells were treated with 10 nM OA, with DMSO or transfected with HRSL3 or the empty vector. Phospho-PKC or total PKC were measured by western blotting using the appropriate antibodies. (D) Twelve hours prior to transfection, OVCAR-3 cells were treated with the myristoylated PKC-specific pseudosubstrate. Then the cells were transfected with HRASL3 expression plasmid and caspase cleavage was analyzed 48 hours after transfection.