To test whether H89 treatments induce apoptosis and the extent CARP-1 is involved in growth inhibitory signaling by H89, we first determined growth inhibition of HBC and COS-7 cells by this agent. Exposure to H89 inhibited cell growth (Figure 1A), in part, by enhancing apoptosis (Figure 1B). Since H89 functions as an antagonist of the PKA catalytic subunit (PKA-C), as well as inhibits MSK1 and ribosomal S6Kinase [14], we next determined whether H89 signaling targeted these kinases to elicit cell growth inhibition. For this purpose, we knocked-down each of these kinases by utilizing on-target-plus Si-RNAs. Western blot analyses of the HBC cells transfected separately with respective Si-RNAs revealed a greater than 70% to almost complete loss of PKA-Cα, S6K, or MSK1 expression when compared with corresponding scrambled Si-RNA-transfected controls, and the targeted depletion of either of these kinases failed to induce apoptosis (not shown). These preliminary findings raise the possibility that different perhaps novel signaling pathway(s) are targeted by H89 to inhibit cell growth.

Whether apoptosis signaling by H89 involves CARP-1 was investigated next. CARP-1 levels were elevated in cells that were treated with H89 or piceatannol (Figure 2A). Studies from our laboratory as well as by others have revealed that CARP-1 is a phosphoprotein [4,16]. We further investigated whether H89 signaling targeted CARP-1 phosphorylation. HBC cells were either untreated or treated with H89, and the CARP-1 protein was immunoprecipitated with anti-CARP-1 (α2) polyclonal antibodies [3], followed by western blot analysis with anti-phospho-tyrosine or anti-phospho-threonine antibodies. The data revealed that while H89 did not cause elevated tyrosine-phosphorylation of CARP-1, the level of threonine phosphorylated CARP-1 was elevated following exposure to H89 (Figure 2B). Thus, H89 treatments cause increased CARP-1 expression as well as its threonine phosphorylation.

The role of CARP-1 in regulating H89-dependent apoptosis was further examined by utilizing multiple MDA-MB-468 HBC sublines that stably express CARP-1 antisense as in methods. Wild-type HBC cells or CARP-1 antisense transfected sublines 1, 4, 9, and 10 were either untreated or treated with H89, and cell lysates analyzed for apoptosis as in methods. As shown in Figure 2C, H89-dependent apoptosis was inhibited in cells that had reduced levels of CARP-1 (sublines 9, 10) when compared to those with normal levels of CARP-1 (wild-type, and sublines 1, 4). Taken together, these results demonstrate that H89-dependent inhibition of HBC cell growth is likely independent of its targeting the PKA, S6K, or MSK1 kinases, while involves elevated threonine phosphorylation and expression of CARP-1. Moreover, since depletion of CARP-1 interferes with apoptosis by H89, our data further underscore an important role for CARP-1 in signaling by H89.

We have previously noted that expression of 14-3-3/stratifin interferes with apoptosis by CARP-1, and that CARP-1 interacts with 14-3-3/stratifin [3]. Moreover, 14-3-3 was also found to bind with TAZ protein in a manner dependent on TAZ serine phosphorylation [8]. Together with the available GenBank database information indicating CARP-1 as a TAZ-binding protein, we speculated that CARP-1 may be part of a multi-protein cytoplasmic complex, and that CARP-1 interactions with 14-3-3 and/or TAZ regulate signaling for cell growth. To test this possibility, we investigated whether CARP-1 interacts with TAZ, the molecular basis of their interaction and the extent these interaction regulates apoptosis. In the first instance, the cell lysates derived from MDA-MB-468 and SKBR-3 breast cancer cells were subjected to immunoprecipitation with anti-CARP-1 (α2) polyclonal antibodies [3] followed by western immunoblotting with anti-TAZ antibodies. As shown in figure 3A, TAZ protein was detected in precipitates derived from anti-CARP-1 antibodies but not from anti-Gst-tag antibodies, suggesting specific interaction between cellular CARP-1 and TAZ proteins. We further ascertained CARP-1 interaction with TAZ by utilizing HCT-116 colon cancer cells that stably express myc-His-tagged wild-type CARP-1. These cells were transfected with plasmids encoding flag-tagged wild-type TAZ (pEF-TAZ-N-Flag) or mutant TAZ proteins (pEF-TAZ(-4)-N-Flag and pEF-TAZ(S89A)-N-Flag that encode TAZ lacking PDZ binding and serine phosphorylation sites, respectively). The TAZ proteins from respective cell lysates were immunoprecipitated with anti-Flag-tag or anti-Gst-tag antibodies, and the immunoprecipitates subjected to western blot analysis with anti-myc-tag antibody. The data revealed the presence of CARP-1 in the immunoprecipitates derived from anti-Flag-tag (lanes 2, 4, and 6) but not from anti-Gst-tag (lanes 1, 3, and 5) antibodies (Figure 3B), suggesting that CARP-1 binding with TAZ does not require the PDZ motif or serine at position 89 of TAZ. Whether TAZ plays a role in CARP-1-dependent apoptosis was investigated next. HBC cells were transfected with vector plasmid pEF1 or pEF1-TAZ-N-Flag followed by incubation of cells with retroviruses encoding vector or myc-His-tagged CARP-1 for a period of 48 h. CARP-1 transduction of cells expressing vector plasmid resulted in ~3-fold increase in apoptosis when compared with their vector transduced counterparts, whereas expression of TAZ inhibited apoptosis induced by CARP-1 (Figure 3C). Since TAZ shuttles between cytoplasmic and nuclear compartments [8] and CARP-1 is a perinuclear protein [3], for their binding to occur, we ascertained the extent the two proteins co-localize. To address this issue, the COS-7 cells were transfected with a combination of plasmids encoding Flag-tagged wild-type TAZ and myc-His-tagged wild-type CARP-1, followed by immunocytochemical detection of fluorescently-labelled respective tagged proteins. As shown in Figure 3D, both the proteins localized predominantly in the cytoplasmic compartment with punctuate nuclear presence.

To map TAZ-interacting domain of CARP-1, cells were transfected with plasmid encoding flag tagged TAZ in combination with the vector plasmid or recombinant plasmids encoding various myc-His-tagged CARP-1 mutants that have been described before [4]. Cell lysates were utilized to immunoprecipitate CARP-1 proteins using anti-myc-tag antibodies followed by western blotting in conjunction with anti-flag tag antibody. The data revealed that TAZ-interacting epitope is present within CARP-1 amino acids 651-759 (Additional file 1A). Similar experimental strategy was utilized to map CARP-1-interacting epitope of TAZ by expressing myc-His-tagged CARP-1 (651-759) mutant in combination with various plasmids encoding gst-tagged TAZ proteins. We found that CARP-1 interacted with a novel epitope within the N-terminal of TAZ, and that TAZ serine 89, WW, PDZ or coiled-coil motifs were not involved in its binding with CARP-1 (Additional file 1B). Our mapping studies are summarized in figure 4, and together with the data in figures 2 and 3 demonstrate that novel motifs of CARP-1 and TAZ are involved in their binding, and that TAZ expression interefers with apoptosis signaling by CARP-1.

To define mechanisms of H89-dependent apoptosis, we first determined whether TAZ-interacting CARP-1 (651-759) mutant is a target of H89 signaling. To address this issue, cells expressing myc-His-tagged CARP-1 (651-759) protein were either untreated or treated with H89. The protein complex was immunoprecipitated with anti-phospho-threonine antibodies, followed by western blotting with anti-myc-tag antibodies. H89 induced threonine phosphorylation of CARP-1 (651-759) protein when compared with the untreated control (Figure 5A) indicating that H89 signaling targets threonine residue(s) within CARP-1 (651-759) protein. Since CARP-1 (651-759) mutant also harbors TAZ-binding epitope (figure 4), we next determined whether threonine phosphorylation of CARP-1 (651-759) regulates CARP-1 binding with TAZ. On the basis of Prosite database analysis [17] that predicted CARP-1 threonine⁶⁶⁷ as a high probability phosphorylation site, we generated additional constructs for expression of CARP-1 (651-759) protein with threonine⁶⁶⁷ substituted to either alanine (T667A) or aspartate (T667D) as detailed in methods. Cells expressing Gst-TAZ (1-120) in combination with myc-His-tagged CARP-1 (651-759), CARP-1 (651-759^T667A), or CARP-1 (651-759^T667D) proteins were utilized to analyze protein complexes that were immunoprecipitated with anti-Gst-tag antibodies followed by western blotting with anti-myc-tag antibodies. The data revealed that TAZ binding with CARP-1 (651-759^T667A) protein was attenuated (Figure 5B, lane 3). T667D substitution in CARP-1 (651-759) protein, on the other hand, enhanced its binding with TAZ (Figure 5B, lane 4) when compared with binding of CARP-1 (651-759) with TAZ. Since substitution of phospho-threonine or phospho-serine residue(s) to aspartate often mimics phosphorylated state of a protein, it is likely that threonine⁶⁶⁷ to aspartate change allows a threonine phosphorylated conformation of CARP-1 (651-759), and favors its binding with TAZ.

Whether CARP-1 threonine⁶⁶⁷ regulates apoptosis signaling was investigated next. Cells were either untransduced or transduced with viruses encoding vector, myc-His-CARP-1 (651-759), or myc-His-CARP-1 (651-759^T667A) proteins for 48 h followed by apoptosis determination as in methods. The cells transduced with myc-His-CARP-1 (651-759) displayed significantly enhanced apoptosis when compared to the apoptosis noted for their vector-transduced or untransduced counterparts (Figure 5C). Expression of myc-His-CARP-1 (651-759^T667A) protein, on the other hand, resulted in lower levels of apoptosis when compared to apoptosis noted in myc-His-CARP-1 (651-759) transduced cells (Figure 5C). These findings suggest that expression of myc-His-CARP-1 (651-759) protein, like wild-type or CARP-1 (603-898) mutant [4] promotes apoptosis while T667A substitution interferes with this function.

Since H89 treatments caused elevated CARP-1 expression and apoptosis, while CARP-1 interacts with TAZ, and TAZ regulates CARP-1-dependent apoptosis (figures 2, 3), we wished to determine whether and to what extent TAZ regulates H89-dependent apoptosis. For this experiment, we first transduced the HBC cells with viruses encoding vector or mouse TAZ (wt) protein for a period of 12 h. The cells were then either untreated or treated with H89 for additional 48 h, followed by determination of apoptosis. Although, exposure to H89 caused statistically significant increase in apoptosis of vector transduced cells, it failed to promote apoptosis in TAZ-transduced cells (Figure 5D). These findings suggest that TAZ interferes with apoptosis signaling by H89, and together with the data in figures 2, 3 and 4 indicate that CARP-1 threonine phosphorylation following H89 treatment promotes TAZ sequestration by CARP-1 that, in part, contributes to CARP-1-dependent growth suppression by H89.

Since we previously found that CARP-1 negatively regulates c-myc expression [3], we wished to determine whether and to what extent expression of cell proliferation regulatory genes such as c-myc and topoisomerase IIα are a target of CARP-1-mediated H89 signaling and the mechanisms involved therein. To test this possibility, we first determined whether expression of c-myc and topoisomerase IIα is inhibited by CARP-1 and its TAZ-interacting mutants. Expression of wild-type CARP-1 or its TAZ-interacting mutants {CARP-1 (603-898) or CARP-1 (651-759) proteins} resulted in reduced levels of c-myc as well as topoisomerase IIα when compared with their vector-transduced counterparts (Figure 6A). Furthermore, treatments with H89 or piceatannol also resulted in diminished c-myc expression in HBC cells when compared with their untreated counterparts (Figure 6B). Since, H89 causes elevated expression of CARP-1 and apoptosis, the findings in figure 6A and 6B suggest that cell growth inhibition following H89 treatments are due, in part, to CARP-1-dependent loss of cell growth promoting factors such as c-myc.

In the next set of experiments, we investigated the mechanism(s) of H89-dependent loss of c-myc expression. Since TAZ is a transcriptional co-activator and CARP-1 is also a regulator of nuclear receptor complex [6,8], we first examined whether CARP-1 induction following H89 treatment attenuates c-myc transcription. To this end, we utilized a plasmid where luciferase reporter is driven by a mouse c-myc promoter from positions -1138 to +580 relative to the P1 transcription start site [18,19]. As expected, significant loss of c-myc promoter activity was noted in H89-treated HBC cells (Figure 6C, top left). The extent of CARP-1 attenuated c-myc transcription was further investigated by transducing c-myc promoter-luciferase reporter-transfected HBC cells with retroviruses encoding vector, wild-type CARP-1, or CARP-1 (651-759) proteins. Expression of wild-type CARP-1 or its TAZ-interacting 651-759 mutant caused significant loss of c-myc promoter activity when compared with their vector-transduced counterparts (Figure 6C, top right). Additional transfection studies revealed that CARP-1/TAZ-responsive element(s) is located within -140 to +310 region of the mouse c-myc promoter (Figure 6C, lower). Taken together, data in figure 6 demonstrate that attenuation of c-myc expression by H89 or CARP-1 is accomplished, in part, by transcriptional down-regulation of c-myc, and contributes to the growth suppressing effects of CARP-1 and H89.

DMEM, Ham's F-12 medium, fetal bovine serum (FBS), and lipofectamine-based transfection kit were purchased from Life Technologies, Inc., Grand Island, NY. Piceatannol, H89, R_p-cAMPS (a competitive antagonist of the cyclic nucleotide-binding domain on PKA), and S_p-cAMPS (a competitive agonist of the cyclic nucleotide-binding domain on PKA) were obtained from Calbiochem/EMD Biosciences (San Diego, CA). The anti-PKA Cα, anti-phospho-PKA C (Thr197) polyclonal antibodies, anti-MSK1, anti-ribosomal S-6-kinase, as well as anti-myc-tag mouse monoclonal (9B11) antibodies were purchased from Cell Signaling (Beverley, MA). Anti-flag M2 and anti-c-myc (clone 67P05) monoclonal antibodies were obtained from Sigma-Aldrich, (St. Louis, MO) and Lab Vision (Fremont, CA), respectively. Anti-phospho-tyrosine and anti-phospho-serine antibodies were purchased from Zymed labs (San Francisco, CA), while Anti-phospho-threonine (H-2) HRP conjugate were from Santa Cruz Biotech (Santa Cruz, CA). Anti-topoisomerase IIα mouse monoclonal (3F6) antibody was from Novacastra/Vector laboratories, Burlingame, CA, and anti-Gst rabbit polyclonal antibody was purchased from Upstate Biotech, Lake placid, NY. Phospho-enrichment beads were purchased from BD Biosciences (Palo Alto, CA).

The construction of plasmid clone 6.1 expressing myc-His-tagged wild-type CARP-1 as well as clone 1.6 expressing CARP-1 antisense has been described before [3]. The plasmids pEF-TAZ-N-Flag, pEF-TAZ(-4)-N-Flag, and pEF-TAZ(S89A)-N-Flag that encode wild type or mutant TAZ proteins were as detailed before [8]. The plasmids encoding N-terminal gst epitope tagged wild type or mutants TAZ proteins were generated by PCR amplification using mouse TAZ cDNA as template followed by their cloning into the pEBG vector plasmid. The plasmid pEBG-TAZ (wt) encodes Gst-tagged wild-type TAZ, while pEBG-TAZ (1-164), pEBG-TAZ (165-395), and pEBG-TAZ (1-120) encode Gst-tagged truncated mouse TAZ proteins. Of note is that fact that the mutant TAZ (1-120) lacks the WW domain that has been implicated in TAZ interactions with PPXY motif of nuclear transcription factors such as c-Jun [8]. In addition, recombinant plasmids for expression of mutant CARP-1 proteins were generated by a combination of standard PCR amplification and cloning methodologies to generate constructs that encode CARP-1 proteins having myc and His tags positioned at the carboxyl termini. Constructs encoding CARP-1 (651-759) protein having threonine at position 667 substituted with alanine or aspartate were similarly generated using a combination of PCR amplification and cloning methods. All the recombinant plasmids were sequenced to ascertain validity of the cDNA inserts. The plasmids having mouse c-myc promoter (-1138 to +580, -426 to +580, -140 to +310, and -140 to +175 relative to the P1 transcription site) driving luciferase reporter gene have been described before [18,19].

ER-negative, p53-negative SKBR-3, MDA-MB-468 HBC cells, and their sublines expressing CARP-1 antisense (Clone 1.6 sublines 1, 4, 9, and 10), as well as human colon cancer HCT-116 cells were cultured and maintained as detailed before [3,19]. HCT-116 sublines stably expressing vector plasmid or myc-His-tagged wild type CARP-1 were isolated and characterized as described before [4]. Monkey kidney COS-7 and NIH3T3 mouse fibroblast cells were obtained from ATCC (Manassas, VA) and propagated per vendor's suggestions. NIH3T3 derivative PT-67 mouse fibroblasts were purchased from BD-Clontech (Palo Alto, CA), and generation and characterization of their stable isolates expressing vector pLNCX2, myc-His-tagged wild-type or mutant CARP-1 proteins was carried out as previously described [3,4].

Cells were plated either in a 60-mm dish or in a 6-well plate at a density of 3 × 10⁵ cells/ml and then co-transfected with pTK/Renilla Luc in combination with various c-myc-Promoter-luc constructs essentially following previously detailed methods [3,4,25]. After 6-h incubation with plasmid DNAs, cells were washed three times with 1 × PBS, co-incubated with retroviruses encoding vector, wild-type or mutant CARP-1 proteins, and allowed to grow in the appropriate culture media for additional 48 h. The cells were then harvested, lysed, and Renilla and firefly luciferase activities were measured using dual luciferase assay kit (Clontech) essentially following vendors guidelines.

CARP-1 expression was down-regulated in HBC cells by stably expressing a plasmid expressing CARP-1 antisense cDNA. Antisense transfected sublines 1, 4 express normal levels, while sublines 9 and 10 express 50% reduced levels of CARP-1 and their characterization has been detailed before [3]. Depletion of PKA-Cα, CARP-1, S6K, or MSK1 was carried out by transfecting MDA-MB-468 HBC cells with scrambled (non-target) or respective specific on-target Si-RNAs (Dharmacon) following manufacturer suggested guidelines. Seventy-two hours post-transfections, the cell lysates were analyzed by western immunoblotting in conjunction with respective antibodies essentially following the methodologies detailed below.

Logarithmically growing cells were either untransfected or transfected with various plasmids for 24 or 48 hours followed by their lysis to prepare protein extracts. In certain instances cells were treated with H89. For immunoprecipitation aliquots of cell lysates containing 1.0 mg proteins were incubated with indicated antibodies and Sepharose G at 4°C for 3 hours. Immunoprecipitates or the protein extracts were then analyzed by western blotting using indicated antibodies per the manufacturer's guidelines and previously published protocols [3,4,19]. For enrichment of phosphorylated proteins, the cells were first transfected with plasmids encoding wild-type or mutant CARP-1 proteins, followed by treatments of cells with H89. Equal amounts (1 mg) of cell lysates were then applied to phospho-protein enrichment columns; the complexes were washed and eluted following manufacturer suggested methodology. The enriched phospho-proteins alongwith the protein lysate from transfected cells were analyzed by western blotting first with anti-myc tag antibodies followed by reprobing with anti-phospho-tyrosine antibodies. In addition, cell lysates were prepared from untreated as well as treated cells to determine apoptosis by utilizing cell death detection ELISA kit (Roche Diagnostics). The net absorbances at the suggested wavelengths (A₄₀₅nm minus A₄₉₅nm) were obtained for the lysates derived from treated as well as untreated cells. The histograms indicating levels of apoptosis were generated either by plotting net absorbances or by calculating the "enrichment factor" as described [3,4]. In certain instances, cell death was measured by counting acridine orange-ethidium bromide stained cells [25], while viabilities of untreated and treated cells were also measured by utilizing LIVE/DEAD viability/cytotoxicity assay (Molecular Probes, Eugene, OR).

COS-7 cells were transfected with plasmid expressing myc-His-tagged wild-type CARP-1 (CARP-1 clone 6.1; ref. [3]) in combination with plasmid expressing wild-type TAZ (pEF/mTAZ-N-Flag, ref. [8]). The cells were trypsinized, fixed, and permeablizied followed by incubation with a combination of anti-Flag tag mouse monoclonal and anti-myc tag rabbit polyclonal antibodies at 4°C for overnight. The cells were washed again in 1 × PBS for three times, and incubated with a combination of rhodamine conjugated goat-anti-rabbit and Alexa 488 conjugated goat-anti-mouse IGg for a period of 30 min at 37°C. The cells were washed twice with pre-warmed 1 × PBS, and coverslips were mounted with an aqueous-based anti-fade medium. The cell fluorescence was detected by an Olympus BX51 fluorescent microscope fitted with a triple DAPI/FITC/TRITC cube, and the images were captured by a DP70 digital camera and image acquisition software. The exposure time and magnification (400×) were maintained throughout the capture of images. The images were subsequently analyzed by Image analysis and Adobe Photoshop software.