Phosphorylation of Par-4 by protein kinase A is critical for apoptosis

Abstract

Despite distinct dissimilarities, diverse cancers express several common protumorigenic traits. We present here evidence that the proapoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated protein kinase A (PKA) activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC (selective for apoptosis induction in cancer cells) domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide-, or small interfering RNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA- and phosphorylated T155-dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a procancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.

FIG. 1.

T155 is essential for the apoptotic function of Par-4. (A) Schematic representation of NLS1, NLS2, S154, T155, and the SAC domain of full-length Par-4. PC-3 cells were transiently transfected with vector, Par-4, ΔNLS2, or the point mutant 154A or 155A; expression of Par-4 and mutants was examined by Western blot analysis with the Par-4 antibody or actin antibody (B). The ability of Par-4 or the mutants to induce apoptosis at 48 h posttransfection (C and D) was examined in PC-3 cells by either ICC with the Par-4 antibody and secondary antibody conjugated to Alexa Fluor 488 (green fluorescence) followed by nuclear staining with DAPI (D, left) or by staining with FITC-conjugated annexin V antibody (D, right). Cells expressing Par-4 or mutant proteins were scored for chromatin condensation or annexin V-positive cells indicative of apoptosis by confocal microscopy, as described in Materials and Methods. (C) Quantitative data. Nomarski images show total number of cells in each field (D, right). To determine inhibition of NF-κB transcriptional activity by Par-4 or its mutants (E), PC-3 cells were transfected for 48 h with NF-κB-luc reporter construct and β-galactosidase plasmid together with vector, Par-4, or mutant constructs. Whole-cell lysates were subjected to luciferase assays, and luciferase activity was normalized to the corresponding β-galactosidase activity. PC-3 cells were transiently transfected with expression constructs for GFP-SAC or GFP-SAC/155A mutant. Expression of the mutants was confirmed by Western blot analysis with the Par-4 or actin antibody (F), and the ability of the mutants to induce apoptosis was quantified by confocal microscopy (G and H). In the results shown in panel H, DAPI is pseudocolored red, and the yellow fluorescence resulting from the overlay with the GFP fusion proteins indicates nuclear localization. NIH 3T3/Ras cells were transiently transfected with the indicated expression constructs, and the ability of the constructs to induce apoptosis was quantified by confocal microscopy (I and J).

FIG. 1.

T155 is essential for the apoptotic function of Par-4. (A) Schematic representation of NLS1, NLS2, S154, T155, and the SAC domain of full-length Par-4. PC-3 cells were transiently transfected with vector, Par-4, ΔNLS2, or the point mutant 154A or 155A; expression of Par-4 and mutants was examined by Western blot analysis with the Par-4 antibody or actin antibody (B). The ability of Par-4 or the mutants to induce apoptosis at 48 h posttransfection (C and D) was examined in PC-3 cells by either ICC with the Par-4 antibody and secondary antibody conjugated to Alexa Fluor 488 (green fluorescence) followed by nuclear staining with DAPI (D, left) or by staining with FITC-conjugated annexin V antibody (D, right). Cells expressing Par-4 or mutant proteins were scored for chromatin condensation or annexin V-positive cells indicative of apoptosis by confocal microscopy, as described in Materials and Methods. (C) Quantitative data. Nomarski images show total number of cells in each field (D, right). To determine inhibition of NF-κB transcriptional activity by Par-4 or its mutants (E), PC-3 cells were transfected for 48 h with NF-κB-luc reporter construct and β-galactosidase plasmid together with vector, Par-4, or mutant constructs. Whole-cell lysates were subjected to luciferase assays, and luciferase activity was normalized to the corresponding β-galactosidase activity. PC-3 cells were transiently transfected with expression constructs for GFP-SAC or GFP-SAC/155A mutant. Expression of the mutants was confirmed by Western blot analysis with the Par-4 or actin antibody (F), and the ability of the mutants to induce apoptosis was quantified by confocal microscopy (G and H). In the results shown in panel H, DAPI is pseudocolored red, and the yellow fluorescence resulting from the overlay with the GFP fusion proteins indicates nuclear localization. NIH 3T3/Ras cells were transiently transfected with the indicated expression constructs, and the ability of the constructs to induce apoptosis was quantified by confocal microscopy (I and J).

FIG. 2.

PKA phosphorylates Par-4 at T155. To determine if Par-4 is a phosphorylated protein (A), NIH 3T3/Ras cells were transiently trasfected with vector or Par-4 for 12 h, incubated in phosphate-free medium overnight, and then metabolically labeled with 200 μCi of [³²P]orthophosphate for 5 h. Whole-cell extracts were subjected to immunoprecipitation with Par-4 or control IgG antibody, resolved by SDS-PAGE, and transferred to a PVDF membrane. The blot was autoradiographed (top) and finally probed with the Par-4 antibody (bottom). To determine whether PKA phosphorylates Par-4 at T155 (B), NIH 3T3 cells were transiently transfected with vector, Par-4, 154A, or 155A expression construct for 24 h, and whole-cell extracts were immunoprecipitated with the Par-4 antibody or IgG control antibody. The immunoprecipitated proteins were subjected to an in vitro phosphorylation reaction with purifed PKA enzyme and [γ-³²P]ATP, and then the radiolabeled proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. The blot was finally probed with Par-4 antibody to determine Par-4 or mutant protein levels. Following densitometric scanning of the blots, the amount of ³²P label incorporated into ectopic Par-4 or mutant protein by PKA was normalized to the corresponding protein level detected on the Western blots, and the relative amount of ³²P label incorporated (i.e., PKA phosphorylation) is shown at the bottom of the panel (B). In experiments aimed at determining whether PKA phosphorylates Par-4 in vivo (C), we used 137-187 and 137-187/155A expression constructs, which were first tested for expression by transient transfection, followed by Western blot analysis with Par-4 or actin antibody (C, right). To examine whether PKA phosphorylates Par-4 in vivo (C, left), NIH 3T3/Ras cells were transiently transfected with a vector or 137-187 or 137-187/155A mutant expression constructs in the presence or absence of PKA-inhibitory peptide PKI for 24 h, then metabolically labeled, and immunoprecipitated with the Par-4 antibody or IgG control antibody. The immunoprecipitates were subjected to SDS-PAGE, transferred to nylon membranes, and autoradiographed (C, top left). Finally, the blots were probed with the Par-4 antibody (C, bottom left), as described above (A and B). To characterize the phospho-T155 (pT155) antibody, MEFs were transfected with GFP, GFP-Par-4, or GFP-155A plasmids with or without the PKAc expression construct for 24 h, and Western blot analysis for phospho-T155, Par-4, the PKA catalytic subunit, and actin was performed (D). NIH 3T3/Ras (E) or PC-3 (F) cells were transiently transfected with vector, Par-4, 154A, or 155A plasmids (E and F, left), or with GFP, GFP-Par-4, or GFP-Par-4/155A plasmid; the cells were then treated for 24 h with vehicle or 20 or 40 μM PKI (E and F, right) and then subjected to Western blot analysis with pT155, Par-4, or actin antibodies. To determine whether inhibition of PKA activity inhibits apoptosis by Par-4 (G), PC-3, NIH 3T3/Ras, or MDA MB 231 cells were transiently transfected with GFP or GFP-Par-4. The PC-3 transfectants were treated with vehicle or with 20 or 40 μM PKI; NIH 3T3/Ras or MDA MB 231 cells were treated with 20 μM PKI. The transfectants and cells were then assayed for apoptosis, as described in the legend to Fig. 1. To ascertain that inhibition of PKA activity by PKI inhibits apoptosis by the SAC domain in diverse cancer cell lines, the cells were transfected with the SAC expression construct, treated with 20 μM PKI peptide or control, and scored for apoptosis (H). To confirm that PKI inhibits PKA activity, PC-3, NIH 3T3/Ras, or MDA MB 231 cells were treated with control or 20 μM PKI peptide for 48 h, and whole-cell lysates were tested for PKA activity with the cAMP-dependent PKA Signatect assay kit (I).

FIG. 2.

PKA phosphorylates Par-4 at T155. To determine if Par-4 is a phosphorylated protein (A), NIH 3T3/Ras cells were transiently trasfected with vector or Par-4 for 12 h, incubated in phosphate-free medium overnight, and then metabolically labeled with 200 μCi of [³²P]orthophosphate for 5 h. Whole-cell extracts were subjected to immunoprecipitation with Par-4 or control IgG antibody, resolved by SDS-PAGE, and transferred to a PVDF membrane. The blot was autoradiographed (top) and finally probed with the Par-4 antibody (bottom). To determine whether PKA phosphorylates Par-4 at T155 (B), NIH 3T3 cells were transiently transfected with vector, Par-4, 154A, or 155A expression construct for 24 h, and whole-cell extracts were immunoprecipitated with the Par-4 antibody or IgG control antibody. The immunoprecipitated proteins were subjected to an in vitro phosphorylation reaction with purifed PKA enzyme and [γ-³²P]ATP, and then the radiolabeled proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. The blot was finally probed with Par-4 antibody to determine Par-4 or mutant protein levels. Following densitometric scanning of the blots, the amount of ³²P label incorporated into ectopic Par-4 or mutant protein by PKA was normalized to the corresponding protein level detected on the Western blots, and the relative amount of ³²P label incorporated (i.e., PKA phosphorylation) is shown at the bottom of the panel (B). In experiments aimed at determining whether PKA phosphorylates Par-4 in vivo (C), we used 137-187 and 137-187/155A expression constructs, which were first tested for expression by transient transfection, followed by Western blot analysis with Par-4 or actin antibody (C, right). To examine whether PKA phosphorylates Par-4 in vivo (C, left), NIH 3T3/Ras cells were transiently transfected with a vector or 137-187 or 137-187/155A mutant expression constructs in the presence or absence of PKA-inhibitory peptide PKI for 24 h, then metabolically labeled, and immunoprecipitated with the Par-4 antibody or IgG control antibody. The immunoprecipitates were subjected to SDS-PAGE, transferred to nylon membranes, and autoradiographed (C, top left). Finally, the blots were probed with the Par-4 antibody (C, bottom left), as described above (A and B). To characterize the phospho-T155 (pT155) antibody, MEFs were transfected with GFP, GFP-Par-4, or GFP-155A plasmids with or without the PKAc expression construct for 24 h, and Western blot analysis for phospho-T155, Par-4, the PKA catalytic subunit, and actin was performed (D). NIH 3T3/Ras (E) or PC-3 (F) cells were transiently transfected with vector, Par-4, 154A, or 155A plasmids (E and F, left), or with GFP, GFP-Par-4, or GFP-Par-4/155A plasmid; the cells were then treated for 24 h with vehicle or 20 or 40 μM PKI (E and F, right) and then subjected to Western blot analysis with pT155, Par-4, or actin antibodies. To determine whether inhibition of PKA activity inhibits apoptosis by Par-4 (G), PC-3, NIH 3T3/Ras, or MDA MB 231 cells were transiently transfected with GFP or GFP-Par-4. The PC-3 transfectants were treated with vehicle or with 20 or 40 μM PKI; NIH 3T3/Ras or MDA MB 231 cells were treated with 20 μM PKI. The transfectants and cells were then assayed for apoptosis, as described in the legend to Fig. 1. To ascertain that inhibition of PKA activity by PKI inhibits apoptosis by the SAC domain in diverse cancer cell lines, the cells were transfected with the SAC expression construct, treated with 20 μM PKI peptide or control, and scored for apoptosis (H). To confirm that PKI inhibits PKA activity, PC-3, NIH 3T3/Ras, or MDA MB 231 cells were treated with control or 20 μM PKI peptide for 48 h, and whole-cell lysates were tested for PKA activity with the cAMP-dependent PKA Signatect assay kit (I).

FIG. 3.

Cancer cells have elevated PKA activity levels. Whole-cell lysates were prepared from various normal or immortalized or cancer cell lines, and an in vitro PKA enzymatic assay was performed with the cAMP-dependent PKA Signatect Assay kit (A to D, left). The cell lines were transiently transfected with vector, GFP-SAC, or GFP-SAC/155A constructs and the transfectants were visualized by confocal microscopy for GFP fluorescence of Par-4 or mutant constructs and DAPI staining for apoptosis (A to D, right). To determine if Par-4 was preferentially phosphorylated in cancer cells compared to normal cells, whole-cell lysates from prostate cancer cell lines LNCaP, LNCaP/IGFBP5, and PC-3 and immortalized prostate epithelial cell line PZ were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with phospho-T155 antibody, Par-4 antibody, and actin antibody as a loading control (E).

FIG. 3.

Cancer cells have elevated PKA activity levels. Whole-cell lysates were prepared from various normal or immortalized or cancer cell lines, and an in vitro PKA enzymatic assay was performed with the cAMP-dependent PKA Signatect Assay kit (A to D, left). The cell lines were transiently transfected with vector, GFP-SAC, or GFP-SAC/155A constructs and the transfectants were visualized by confocal microscopy for GFP fluorescence of Par-4 or mutant constructs and DAPI staining for apoptosis (A to D, right). To determine if Par-4 was preferentially phosphorylated in cancer cells compared to normal cells, whole-cell lysates from prostate cancer cell lines LNCaP, LNCaP/IGFBP5, and PC-3 and immortalized prostate epithelial cell line PZ were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with phospho-T155 antibody, Par-4 antibody, and actin antibody as a loading control (E).

FIG. 4.

Elevation of PKA activity in normal cells activates the apoptotic potential of the SAC domain of Par-4 in a T155-dependent manner. MEF cells were transiently transfected with vector, GFP-Par-4, GFP-SAC, or GFP-SAC/155A expression constructs, treated with either vehicle or 8-Cl-cAMP (10 μM) for 48 h, and examined for intracellular localization of Par-4 or mutants (A) or apoptosis (B). MEF cells were cotransfected with expression constructs for GFP-Par-4, GFP-SAC, or vector and PKAc for 48 h; the transfected cells were visualized under a confocal microscope by GFP fluorescence for Par-4 or mutants or by immunostaining with PKAc antibody, followed by Texas red-conjugated secondary antibody, and for apoptosis by DAPI staining (C). Apoptotic cells were scored and the data were presented as percent apoptosis (D, left). PKA activity was determined with the cAMP-dependent PKA Signatect assay kit (D, right). Protein expression was examined by Western blot analysis with antibodies for GFP, PKAc, or actin (E).

FIG. 4.

Elevation of PKA activity in normal cells activates the apoptotic potential of the SAC domain of Par-4 in a T155-dependent manner. MEF cells were transiently transfected with vector, GFP-Par-4, GFP-SAC, or GFP-SAC/155A expression constructs, treated with either vehicle or 8-Cl-cAMP (10 μM) for 48 h, and examined for intracellular localization of Par-4 or mutants (A) or apoptosis (B). MEF cells were cotransfected with expression constructs for GFP-Par-4, GFP-SAC, or vector and PKAc for 48 h; the transfected cells were visualized under a confocal microscope by GFP fluorescence for Par-4 or mutants or by immunostaining with PKAc antibody, followed by Texas red-conjugated secondary antibody, and for apoptosis by DAPI staining (C). Apoptotic cells were scored and the data were presented as percent apoptosis (D, left). PKA activity was determined with the cAMP-dependent PKA Signatect assay kit (D, right). Protein expression was examined by Western blot analysis with antibodies for GFP, PKAc, or actin (E).

FIG. 5.

Endogenous Par-4 induces T155- and PKA-dependent apoptosis. To study localization of Par-4 and apoptosis in response to cAMP and doxorubicin treatment (A and C), MEFs were treated with vehicle, 10 μM 8-Cl-cAMP, 100 nM doxorubicin, 8-Cl-cAMP plus doxorubicin, or 8-Cl-cAMP plus 20 μM PKI and subjected to ICC analysis with Par-4 antibody followed by FITC-conjugated secondary antibody (green). The nuclei were stained with DAPI (pseudocolored red) and visualized under a confocal microscope (A) and scored for apoptosis (A and C). To study phosphorylation of T155 by cAMP (B), MEFs were treated with 10 μM cAMP, cAMP plus PKI 20 μM, or cAMP plus 40 μM PKI; whole-cell lysates were subjected to Western blot analysis with pT155, Par-4, pCREB, total CREB, or actin antibodies (B, top). PKA activity was determined in the lysates with the cAMP-dependent PKA Signatect assay kit (B, bottom). To determine whether vincristine induces T155 phosphorylation of Par-4 (D), HEL cells were treated with vehicle or 100 nM vincristine for 24 and 48 h, and Western blot analysis was performed on the whole-cell lysates with pT155, Par-4, or actin antibodies. To study vincristine-inducible apoptosis (E), HEL cells were treated with vehicle or vincristine in the presence or absence of 20 or 40 μM PKI peptide or transiently transfected with vector or GFP-Par-4 155A plasmid and assayed for apoptosis as described in the legend to Fig. 1. To determine whether inhibition of Par-4 or PKA expression inhibits apoptosis by vincristine (F), HEL cells were transiently transfected with 10 μM nonspecific control siRNA, siRNA for Par-4, or siRNA for PKA α plus β duplexes, treated with either vehicle or 100 nM vincristine, and assayed for apoptosis as described in the legend to Fig. 1. Whole-cell lysates from the siRNA-transfected cells were subjected to Western blot analysis with antibodies against PKAc α and β, Par-4, and actin (G). To determine the effect of the various treatments on PKA activity, whole-cell lysates prepared from the cells after various treatments, as described above (D and E), were tested for PKA enzymatic assay with the cAMP-dependent PKA Signatect assay kit (H).

FIG. 5.

Endogenous Par-4 induces T155- and PKA-dependent apoptosis. To study localization of Par-4 and apoptosis in response to cAMP and doxorubicin treatment (A and C), MEFs were treated with vehicle, 10 μM 8-Cl-cAMP, 100 nM doxorubicin, 8-Cl-cAMP plus doxorubicin, or 8-Cl-cAMP plus 20 μM PKI and subjected to ICC analysis with Par-4 antibody followed by FITC-conjugated secondary antibody (green). The nuclei were stained with DAPI (pseudocolored red) and visualized under a confocal microscope (A) and scored for apoptosis (A and C). To study phosphorylation of T155 by cAMP (B), MEFs were treated with 10 μM cAMP, cAMP plus PKI 20 μM, or cAMP plus 40 μM PKI; whole-cell lysates were subjected to Western blot analysis with pT155, Par-4, pCREB, total CREB, or actin antibodies (B, top). PKA activity was determined in the lysates with the cAMP-dependent PKA Signatect assay kit (B, bottom). To determine whether vincristine induces T155 phosphorylation of Par-4 (D), HEL cells were treated with vehicle or 100 nM vincristine for 24 and 48 h, and Western blot analysis was performed on the whole-cell lysates with pT155, Par-4, or actin antibodies. To study vincristine-inducible apoptosis (E), HEL cells were treated with vehicle or vincristine in the presence or absence of 20 or 40 μM PKI peptide or transiently transfected with vector or GFP-Par-4 155A plasmid and assayed for apoptosis as described in the legend to Fig. 1. To determine whether inhibition of Par-4 or PKA expression inhibits apoptosis by vincristine (F), HEL cells were transiently transfected with 10 μM nonspecific control siRNA, siRNA for Par-4, or siRNA for PKA α plus β duplexes, treated with either vehicle or 100 nM vincristine, and assayed for apoptosis as described in the legend to Fig. 1. Whole-cell lysates from the siRNA-transfected cells were subjected to Western blot analysis with antibodies against PKAc α and β, Par-4, and actin (G). To determine the effect of the various treatments on PKA activity, whole-cell lysates prepared from the cells after various treatments, as described above (D and E), were tested for PKA enzymatic assay with the cAMP-dependent PKA Signatect assay kit (H).

FIG. 6.

Schematic representation of PKA- and T155-dependent apoptosis by Par-4 in normal and cancer cells. Based on our findings, we propose the following model for differential apoptosis by Par-4 in cancer and normal cells. PKA activity, which is constitutively elevated in cancer cells, causes phosphorylation of Par-4 at the T155 residue. The translocation of phospho-T155 to the nucleus results in inhibition of NF-κB activity and apoptosis. By contrast, PKA activity and T155 phosphorylation levels are relatively low in normal cells. In addition, Par-4 translocation to the nucleus is blocked by an unknown mechanism. Consequently, normal cells fail to undergo apoptosis with Par-4. However, proapoptotic stimuli, such as cAMP or vincristine, cause PKA-dependent phosphorylation of T155; doxorubicin or vincristine can cause translocation of Par-4 to the nucleus. Therefore, vincristine alone or a combination of cAMP and doxorubicin can induce apoptosis of the normal cells in a PKA- and T155 Par-4-dependent mechanism. The mechanism of nuclear translocation of Par-4 is currently under investigation.