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J. Biol. Chem., Vol. 276, Issue 50, 47583-47589, December 14, 2001

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Protein Associated with Myc (PAM) Is a Potent Inhibitor of Adenylyl Cyclases^*

Klaus Scholich, Sandra Pierre, and Tarun B. Patel^§¶

From the Department of Pharmacology and the ^§ Vascular Biology Center, University of Tennessee, Memphis, Tennessee 38163

Received for publication, August 14, 2001, and in revised form, September 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Using the yeast two-hybrid assay and the second of the two large cytosolic domains of type V adenylyl cyclase (ACV) as bait, we identified a small region (amino acids 1028-1231) in the protein associated with Myc (PAM) as an interaction site for ACV. This small region of PAM as well as purified full-length PAM inhibited the activity of ACV. Additionally, full-length PAM was a very potent inhibitor of ACI and AC activities in S49 cyc cells and HeLa cells with IC₅₀ values in the pM and low nM range. Moreover, the regulator of chromatin condensation 1-like domain of PAM (amino acids 446-1062) was sufficient and as potent as full-length PAM at inhibiting the activity of ACV. Interestingly, full-length PAM did not inhibit ACII activity that was stimulated by either forskolin of G_s. When endogenous levels of PAM in HeLa cells were decreased using antisense oligodeoxynucleotides, the basal cAMP content was elevated, and the dose-response curve for vasoactive intestinal peptide-elicited cAMP accumulation in HeLa cells was shifted to the left. Therefore, we conclude that PAM is a very potent, novel inhibitor of specific isoforms of AC. Furthermore, the regulator of chromatin condensation 1-like domain of PAM is sufficient to exert the effects of the full-length protein on AC and decreases in endogenous PAM levels in HeLa cells can modulate both basal and agonist stimulated cAMP accumulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Adenylyl cyclase (AC)¹ catalyzes the conversion of ATP into cAMP. Presently, 10 isoforms of this enzyme have been cloned and characterized (for review, see Refs. 1-3). Nine of these isoforms are membrane-bound, and one is soluble (1, 2, 4, 5). The soluble AC is found in cells and tissues in which changes in HCO concentrations alter cAMP levels (5).

Based upon their sequence homologies and regulation by a variety of modulators, the nine membrane-bound isoforms of AC can be divided into four groups (for elaboration, see Ref. 3). Group 1 consists of AC types I (ACI), III (ACIII), and VIII (ACVIII), which are regulated by Ca²⁺ and calmodulin (6-10). ACI and ACVIII are stimulated by Ca²⁺ and calmodulin (6, 8, 9), but ACIII is only activated by Ca²⁺/calmodulin if Gpp(NH)p or forskolin is present (10). However, in intact cells the ACIII is inhibited by Ca²⁺ via the actions of Ca²⁺/calmodulin-dependent protein kinase II (11-13). The second group comprises types II (ACII), IV (ACIV), and VII (ACVII) isoform. These isoforms are stimulated by G subunits of the heterotrimeric G proteins provided that the active  subunit (G_s) of the stimulatory GTP-binding protein G_s is also present (14-17). Types V (ACV) and VI (ACVI) isoform, which are the predominant ACs in the heart (18-21), form the third group. Both of these enzymes are inhibited by the G_i subunit and directly by calcium (18-21). The fourth group consists of an AC isoform (type IX) that is regulated by calcineurin (22). Despite the differences in their regulation, all membrane-bound isoforms of AC are stimulated by the stimulatory GTP-binding protein of AC, G_s (1, 2, 23). Similarly, all isoforms with the exception of ACIX (24, 25) are stimulated by the diterpene, forskolin.

In addition to the modulators described above, activity of the membrane-bound AC isoforms has also been shown to be regulated by other proteins. For instance, peptides corresponding to regions in caveolin 1 and 2 have been shown to inhibit the activity of ACV (26). Similarly, a bacterial protein, cis-trans-peptidylprolyl isomerase, has been demonstrated to inhibit the activity of ACVII and certain other isoforms (27). More recently, ACIII, ACV, and ACVI have been shown to be inhibited by RGS2 (28). In a search for novel mammalian proteins that may interact with ACs and regulate their activity, we performed a two-hybrid yeast screen with the second cytosolic domain of ACV, C2 (29), as a bait. Our screen isolated a clone that corresponds to a region within the protein associated with Myc (PAM (30)). The homologs of PAM in Caenorhabditis elegans (RPM-1) and in Drosophila (HIW) have been shown to be important for synaptogenesis, synaptic growth, and presynaptic organization (31-33). However, the mechanisms of action of PAM at the cellular level remain to be elucidated. Here we show that purified PAM as well as a protein corresponding to its regulator of chromatin condensation 1 (RCC1) homology domain potently inhibit the activity of ACI, ACV, ACVI and ACVII, and AC in HeLa cells without altering the activity of ACII. PAM and its RCC1 domain are more potent inhibitors of AC activity than either G_i or G subunits of heterotrimeric G proteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Yeast Two-hybrid Assay-- The yeast two-hybrid assay was performed using the HF7c yeast strain provided in the Matchmaker^TM kit. Growth conditions, media, and transformation protocols followed the manufacturer's instructions. Yeast cells transformed with plasmid construct C2ACV-pGBT9, which encodes for the C2 domain of ACV (see Ref. 34), were cotransformed with an adult rat brain cDNA library in plasmid pGAD10 (CLONTECH). Approximately 15 million transformed yeast cells were grown on plates containing either medium deficient in L-histidine, L-leucine, and L-tryptophan (Leu/Trp/His) or medium lacking only Leu and Trp. Yeasts growing in the latter medium demonstrate successful transformation with both plasmids, and those growing in His medium suggest interaction between proteins. The plates were incubated at 30 °C for 7 days. Several of the colonies that grew on plates containing Leu/Trp/His medium were then individually streaked out onto new plates containing the same medium for another 3 days. These clones were grown overnight in liquid medium lacking Leu, Trp, and His at 30 °C followed by 3 h in complete medium. The cells were then pelleted, and -galactosidase activity in cell lysates was detected by the chemiluminescence assay of Jain and Magrath (35) as described in our previous publications (34, 36). The cDNAs from clones that showed -galactosidase activity were isolated and sequenced. To control for false positive interactions, the isolated cDNA was used to cotransform yeast with C2ACV-pGBT9 or GBT9 alone. Analysis of the transformants was performed as described above.

Purification of Full-length PAM-- HeLa cells were grown in DMEM with 10% fetal bovine serum and 1% penicillin and streptomycin. Confluent cells from 25 150-mm dishes were harvested by trypsinization and pelleted for 5 min at 400 × g. The cells were resuspended in TED buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol) containing 135 mM NaCl, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM benzamidine, and 5 µg/ml soybean trypsin inhibitor, lysed by 2 × 5 s of sonication, and subsequently homogenized using 15 strokes in a Dounce homogenizer. The homogenate was centrifuged at 27,000 × g for 30 min at 4 °C, and the supernatant was loaded on a Superdex 200pg gel filtration column (Amersham Pharmacia Biotech). Proteins were eluted with TED containing 135 mM NaCl, and the fractions were analyzed by Western blotting. Positive fractions were pooled, loaded on a Q-Sepharose column, and eluted with a gradient of 150-350 mM NaCl in TED. Fractions containing PAM were pooled and the buffer exchanged against the aforementioned TED buffer containing 100 mM NaCl using Centricon 50 (Amicon, Beverly, MA) according to the manufacturer's instructions. The protein was then loaded on a Mono S 5/5 FPLC column (Amersham Pharmacia Biotech) and washed with the loading buffer (100 mM NaCl in TED). The flow-through was collected and applied to a Mono Q 5/5 FPLC column (Amersham Pharmacia Biotech). The protein was eluted with a gradient from 150 to 400 mM NaCl in TED. Positive fractions were pooled and the buffer exchanged against 50 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol using Centricon 50 (Amicon). Then the protein was applied to a hydroxylapatite column (Bio-Rad) and eluted with a gradient of 100-400 mM sodium phosphate, pH 8.0. Positive fractions were pooled and the buffer exchanged against TED. The purified PAM was stored at 80 °C and used within 3 weeks.

Cloning of PAM Subdomains for Expression in Escherichia coli-- The RCC1-like domain (nucleotides 1484-3333; amino acids 446-1062) was subcloned from clone 97HT5 (gift from Dr. Guo, NIH, Bethesda, MD (30)) into pTrcHisA (Invitrogen, Carlsbad, CA) in two steps. First, a DNA fragment was excised using SspI and EcoRI restriction endonucleases. The SspI site was blunted using Klenow polymerase and cloned in BamHI/EcoRI in pTrcHisA. The BamHI site in pTrcHisA was blunted using Klenow polymerase. In the second step, a cDNA fragment was excised from an overlapping clone T25a (also a gift from Dr. Guo) using EcoRI and DraI and cloned in the EcoRI/HindIII-cut plasmid from the first step. The HindIII and DraI sites were blunted using Klenow polymerase.

Expression and Purification of the RCC1-like Domain of PAM-- The recombinant RCC1-like domain of PAM was expressed in the Top10 strain of E. coli. Cells transformed with plasmid constructs encoding the RCC1-like domain were grown in LB medium containing 50 µg/ml ampicillin at 37 °C until they reached an A₆₀₀ of 0.4. Expression of the protein was induced by the addition of 0.1 mM isopropyl-1-thio--D-galactopyranoside. The cells were incubated for 15 h at 23 °C before they were pelleted at 6,000 × g at 4 °C and resuspended in 10 volumes of 50 mM Tris-HCl, pH 8.0, and 20 µg/ml each aprotinin and leupeptin, 1 mM benzamidine, and 5 µg/ml soybean trypsin inhibitor. Lysozyme was added to a final concentration of 0.1 mg/ml and incubated on ice for 30 min before adding 5 mM MgCl₂ and 0.2 mg/ml DNase. After 5 min, the cells were centrifuged at 27,000 × g for 30 min at 4 °C, and the supernatant was batch-bound for 1 h at 4 °C to metal-chelating resin (Talon, CLONTECH). The resin was then poured into a column and washed with 10 column volumes of buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, followed by a second wash with 10 column volumes of buffer containing 50 mM Tris-HCl, pH 8.0, 20 mM NaCl. The proteins were eluted with 3 column volumes of 50 mM Tris-HCl, pH 8.0, 20 mM NaCl, and 125 mM imidazole and applied directly to a Mono Q 5/5 FPLC column. The protein was then eluted with a 100-350 mM NaCl gradient in TED. Fractions containing the proteins were pooled and concentrated in buffer containing TED using Centricon 50 according to the instructions of the manufacturer. The protein was stored at 80 °C and used within a week.

Expression and Purification of Recombinant G_s-- The hexahistidyl-tagged, constitutively active Q213L mutant of G_s (G_s*) was expressed and purified as described in our previous publication (32). To ensure maximal activation of the G_s*, the G protein was incubated with 1 µM GTPS in the presence of 25 mM MgCl₂ for 30 min prior to use in AC activity assays.

Expression of Type I and V AC in Sf9 Cells-- Sf9 cells that were grown to 70-90% confluence in Sf900 II serum-free medium (Life Technologies, Inc.) were infected with the recombinant baculovirus and 60 h later, harvested in phosphate-buffered saline containing 20 µg/ml phenylmethylsulfonyl fluoride, 3 µg/ml leupeptin, 3 µg/ml soybean trypsin inhibitor, 3 µg/ml aprotinin, and 3 mM benzamidine. The cells were lysed in 25 mM Hepes, pH 7.4, 1 mM EGTA, 10% sucrose, and cell membranes were prepared as described by Kassis and Fishman (37). Aliquots were stored at 80 °C until use.

AC Activity Assays-- In experiments with full-length PAM or the recombinant RCC1-like domain of PAM, membranes (15 µg of protein) were incubated for 20 min on ice in the presence or absence of the proteins of interest. This incubation (20 µl final volume) contained 50 mM Tris-HCl, pH 8.0, and a protease inhibitor mix (5 mM benzamidine, 20 µg/ml soybean trypsin inhibitor, 20 µg/ml leupeptin, and 20 µg/ml aprotinin). The preincubated membranes were then transferred into the AC activity reactions. AC activity assays were performed in a volume of 100 µl for 15 min at room temperature in the presence of 5 mM MgCl₂ as described previously (38). 100 nM G_s* or 100 µM forskolin was used to stimulate enzyme activity. Concentrations of PAM and its RCC1-like domain were calculated based upon the theoretical molecular mass (based on amino acid content) of these proteins (510 kDa for PAM and 70 kDa for its RCC1-like domain).

Antisense Oligodeoxynucleotides and cAMP Measurements-- The sequences of the phosphorothioate oligodeoxynucleotides (ODNs) were as follows: sense, 5'-CTGTTCATGCCGGTT-3'; antisense, 5'-AACCGGCATGAACAG-3'; and antisense ODN harboring three mutations (3M-as; mutations are underlined), 5'-AATCCGTATGAACAC-3'. HeLa cells were plated on 35-mm dishes (300,000 cells/dish) and grown in DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin for 24 h. The ODNs (3 µM each) were introduced into the cells by transfections using FUGENE 6 (Roche, Indianapolis, IN) in serum-free medium according to the instructions of the manufacturer. 90 min later the medium was changed with DMEM containing 10% fetal bovine serum and 3 µM ODNs. The cells were then incubated for 20 h followed by an incubation for 4 h in serum-free DMEM before being treated with 100 µM isobutylmethylxanthine for 5 min. Wherever indicated, cells were incubated for an additional 10 min in the presence of different concentrations of vasoactive intestinal peptide (VIP) or serum. The medium was then aspirated, and the cells were treated with 1 N HCl as described previously (39). The cAMP accumulation in the cells was determined by the radioimmunoassay procedure described by Brooker et al. (40). To check the levels of PAM, cells were harvested in Laemmli buffer and analyzed by immunoblots using anti-PAM antibody (gift from Dr. Guo).

Immunofluorescence Staining-- To monitor the distribution of PAM during the M and G₁ phases of the cell cycle, HeLa cells were incubated with 50 ng/ml nocodazole for 24 h. They were then released from the nocodazole block by replacing medium devoid of the drug. Samples were taken 2 h (M phase) and 8 h (G₁ phase) after release, and cell cycle phases were verified by fluorescence-activated cell sorter analysis. In identical and parallel experiments, HeLa cells were grown on glass coverslips in DMEM with 10% fetal bovine serum and 1% penicillin and streptomycin. The cells were fixed in 4% paraformaldehyde in PBS for 10 min and then permeabilized in 0.1% Triton X-100 for another 5 min. The coverslips were blocked for 1 h in 3% bovine serum albumin in PBS and then incubated for 1 h with anti-PAM antibody (1:50 dilution). This was followed by incubation with fluorescein isothiocyanate-labeled goat anti-rabbit antibody in PBS containing 3% bovine serum albumin. The cells were then washed with PBS and mounted. Staining of nuclei was performed using 1 µg/ml propidium iodide in PBS for 5 min.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

To identify novel proteins that may interact and modulate AC activity, initially, we performed a two-hybrid screen with an adult rat brain cDNA library using the C2 domain of ACV as the bait. A positive clone corresponding to amino acids 1028-1231 of PAM (30) in the correct reading frame was isolated (Fig. 1A). This clone (clone 22) was expressed in bacteria as a hexahistidyl-tagged protein and purified. The purified protein was tested for its ability to modulate AC activity. As shown in Fig. 1B, the protein corresponding to clone 22 (amino acids 1028-1231 of PAM) inhibited the activity of the full-length ACV when the enzyme was stimulated by G_s* (Fig. 1B). However, the full-length ACV activity that had been stimulated by forskolin was not altered in the presence of the protein corresponding to clone 22 (Fig. 1B).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Interaction between a short region of PAM (amino acids 1028-1231, clone 22) and the C2 domain of ACV and inhibition of ACV activity by this domain of PAM. Panel A, yeast cells were transformed with cDNA corresponding to clone 22 in plasmid pGAD424 and pGBT9 or with the C2 domain of ACV in pGBT9. The yeast cells were grown in liquid medium lacking His, Leu, and Trp, and -galactosidase activity was monitored as described under "Materials and Methods." The means ± S.E. from at least seven determinations are shown; *p < 0.006, Student's unpaired t test. Panel B, the protein corresponding to the region of PAM encoded by clone 22 (amino acids 1028-1231) was expressed with a hexahistidyl tag and purified. The purified protein was then tested for its activity in AC activity assays with membranes from Sf9 cells expressing the full-length ACV. AC activity was stimulated by the addition of either 100 nM Gs* or 100 µM forskolin. The Gs*- and forskolin-stimulated specific activities of ACV were 46.5 ± 5 and 67.5 ± 6.2 pmol/min/mg, respectively. The means ± S.E. of at least two experiments done in triplicate are represented; *p  0.001; **p < 0.01, Student's unpaired t test analyses.

Next, we investigated whether or not the full-length PAM also modulates AC activity. For this purpose, PAM was purified from HeLa cells as described under "Materials and Methods." Fig. 2A shows the purification of PAM at different steps in the procedure. The final purified protein was a single band on the gel and was estimated to be more than 95% pure (Fig. 2A, last lane). Western analyses of the purified protein in lane 6 of Fig. 2 with anti-PAM antibody showed a single band (Fig. 2B). Using this purified protein, we tested its ability to inhibit AC activity in HeLa cells. As shown in Fig. 2C, PAM inhibited the G_s*-stimulated AC activity in HeLa cell lysates in a concentration-dependent manner such that maximal inhibition was achieved at ~10 nM protein. However, like the findings with the protein corresponding to clone 22 (Fig. 1B), the forskolin-stimulated AC activity in HeLa cell lysates was not altered by full-length PAM. Thus the full-length PAM mimics the actions of the short protein, which corresponds to its amino acids 1028-1231.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Purification of full-length PAM and inhibition of AC activity by PAM in HeLa cells. Panel A, full-length PAM was purified from HeLa cells as described under "Materials and Methods." The PAM-enriched fractions from different stages of purification were subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% acrylamide gel and stained with Coomassie Blue. The numbers above the lanes correspond to the step in the purification procedure shown on the left. M designates the lane in which the molecular mass markers were run. Each lane contains the following amount of protein: lane 1, 40 µg; lane 2, 15 µg; lane 3, 8 µg; lane 4, 4 µg; lane 5, 1.5 µg; and lane 6, 1.0 µg. For the purification presented, the total amount of protein in the HeLa cell lysate was 95 mg, and the final amount of PAM obtained was 145 µg. Typically, each purification yields 150-200 µg of protein. Panel B, Western analyses of the purified PAM protein with anti-PAM antibody. 500 ng of purified PAM protein was separated on a 6.5% polyacrylamide gel. The protein was transferred to nitrocellulose membrane and blotted with anti-PAM antibody (1:500 dilution, 1 h at 37 °C in 3% bovine serum albumin in PBS). Panel C, AC activity of HeLa cell lysates was measured in the absence and presence of the indicated concentrations of the full-length PAM protein as described under "Materials and Methods." The data are presented as a percent of control and are the means ± S.E. of three experiments each done in triplicate. Control Gs- and forskolin-stimulated AC activities were 130 ± 11 and 573 ± 17 pmol/min/mg of protein, respectively.

In additional experiments, we investigated the ability of PAM to modulate the activity of different AC isoforms. As observed with the protein corresponding to clone 22 (Fig. 1B), PAM inhibited the G_s*-stimulated activity of full-length ACV expressed in Sf9 cells. Again, ACV activity that had been stimulated with forskolin was not altered by PAM (Fig. 3A). PAM also inhibited G_s*-stimulated ACI activity but not the forskolin-stimulated activity of this isozyme (Fig. 3B). Interestingly, PAM did not alter either the G_s*- or forskolin-stimulated activity of ACII expressed in Sf9 cells (Fig. 3C). These data demonstrate that PAM differentially modulates the activity of different isoforms. Moreover, the finding that PAM does not inhibit G_s*-stimulated ACII activity indicates that PAM does not alter AC activity by merely sequestering G_s* or blocking the actions of the G protein in some nonspecific manner. Further evidence of differential regulation of the AC isozymes is shown by the finding that in membranes of S49 cyc cells, PAM inhibited both the forskolin- and G_s*-stimulated AC activity. The predominant AC isoforms in S49 cells have been shown to be ACVI and ACVII (41). Thus, unlike ACV, ACI (Fig. 3, A and B), and AC activity in HeLa cells (Fig. 2C), PAM can inhibit the activity of ACVI+VII (S49 cell membranes; Fig. 3D) when the enzyme is stimulated by forskolin. Whether or not PAM inhibits forskolin-stimulated AC activity in other cell types or tissues remains the subject of further investigations. Another interesting aspect of the regulation of AC activity by PAM is the sensitivity of the different isoforms. Hence, for the G_s*-stimulated activities of ACI and ACV the IC₅₀ concentrations of PAM are 0.3 and 3 nM, respectively. In the case of S49 cells (ACVI+VII), PAM inhibits forskolin- and G_s*-stimulated activities with IC₅₀ values of ~0.03 nM. For comparison, G_s*-stimulated ACV and Ca²⁺/calmodulin-stimulated ACI activities are inhibited by G_i with IC₅₀ values of ~100 and 30-100 nM, respectively (42, 43). Likewise, G subunits of G protein also inhibit ACI with IC₅₀ values that are much higher (approximately 50 nM (42)) than those we report here for PAM. Recently, bacterial cis-trans-peptidylprolyl isomerase (27) and RGS2 (28) have been shown to inhibit certain isoforms of AC. However, these proteins also inhibit AC activity at very high concentrations (27, 28). Overall, therefore, PAM is a more potent inhibitor of AC activity than G_i or any other protein that has been shown to inhibit AC activity.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Inhibition of different isoforms of AC by PAM. AC activity of ACV (panel A), ACI (panel B), ACII (panel C), and AC in S49 cyc cells, which is predominantly ACVI and ACVII (panel D) (41), was assayed in the presence of different concentrations of full-length PAM. The activities of the various AC isoforms were stimulated with either 100 nM Gs* (white circles) or 100 µM forskolin (black squares). Data from at least three experiments, each done in triplicate, are presented as a percent of control and are the means ± S.E. The control (100%) activities (all in pmol/min/mg of protein) were: ACV: Gs, 47.4 ± 3.7; forskolin, 47.9 ± 6.2; ACI: Gs, 30.7 ± 1.2; forskolin, 42 ± 2.5; ACII: Gs, 217 ± 19.6; forskolin, 52.1 ± 11.6; S49 cyc : Gs, 111 ± 3.7; forskolin, 159.3 ± 17.5.

Interestingly, PAM contains a region (amino acids 498-1066) that is homologous to the RCC1 protein (30). The crystal structure of the RCC1 protein has been solved recently (44) and resembles the seven-bladed propeller structure of the G protein  subunit (45). Because clone 22 includes the C-terminal end of the RCC1-like domain of PAM and because G protein subunits have been shown to modulate AC activity (for review, see Refs. 1, 2, and 23), we reasoned that the RCC1-like domain of PAM may be the part of the protein which is responsible for the inhibition of AC activity. Therefore, we expressed and purified a protein corresponding to amino acids 446-1062 of PAM which encompasses the RCC1-like domain and compared its activity with full-length PAM. As shown in Fig. 4, PAM and its RCC1-like domain inhibited G_s*-stimulated ACV activity with equivalent potency. Moreover, as shown for PAM (Fig. 3A), the RCC1-like domain of PAM did not alter the forskolin-stimulated ACV activity (not shown). These results demonstrate that the RCC1-like domain of PAM is sufficient to observe inhibition of AC activity.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   The RCC1-like domains of PAM and full-length PAM are equipotent at inhibiting ACV activity. Full-length PAM and its RCC1-like domain were purified as described under "Materials and Methods." These proteins were then added to the AC activity assays with membranes from Sf9 cells expressing ACV as detailed under "Materials and Methods." The means ± S.E. of three determinations are shown. To facilitate comparison, the data for inhibition by full-length PAM from Fig. 3A are shown again.

The RCC1 protein is a seven-bladed propellor and resembles the structural features of the G protein  subunit (44, 45). Because G protein G subunits inhibit ACI activity (46), at first sight it is tempting to speculate that this structural similarity is the basis for the inhibition of ACV activity by the RCC1-like domain. However, this similarity cannot completely explain the actions of PAM on ACV and ACII. Thus, ACV activity is not inhibited in in vitro experiments by G protein subunits (42). Although Bayewitch et al. (47) have shown that in cells overexpressing ACV the enzyme was inhibited by G₁₂, whether or not the effects of G which they observed were direct or indirect remains to be determined. Moreover, ACII activity has been demonstrated to be conditionally stimulated in the presence of G subunits (46). However, we did not observe any stimulation of ACII activity in the presence of PAM and G_s* (Fig. 3C). Therefore, whether or not the structural similarity between the RCC1-like domain and G protein  subunits forms the basis for the inhibitory activity of PAM remains to be determined.

To determine whether PAM modulates AC activity in intact cells, we used the approach of decreasing the amount of endogenous PAM in HeLa cells with antisense ODNs. As shown in Fig. 5A, in HeLa cells treated with antisense ODN the amount of PAM, as determined by Western analysis, was decreased compared with cells treated with sense or mutant antisense ODNs. Reprobing the same blot with anti-epidermal growth factor receptor antibody showed that the loading of proteins was the same (Fig. 5A). The mutant antisense ODN was not totally inactive and decreased the amount of PAM to some extent (Fig. 5A). Partial suppression of activity with mutant antisense ODNs has been observed previously by others (48). Interestingly, the forskolin-stimulated AC activity in lysates of cells treated with PAM was unchanged, indicating that the total amount of AC was not altered by the ODNs (Fig. 5B). However, the treatment of HeLa cells with antisense ODN increased basal cAMP levels (Fig. 5C). Consistent with the partial decrease in the amount of PAM, the levels of cAMP in cells treated with mutant antisense ODN were intermediate between those in cells treated with the sense and antisense ODNs (Fig. 5C). Moreover, when the amount of PAM was decreased by treatment of HeLa cells with antisense ODNs, the dose-response curve of cAMP accumulation in response to VIP was shifted to the left (Fig. 5D). Hence, in the presence of antisense ODN, when the endogenous amount of PAM was low, VIP stimulated cAMP accumulation to the same level at ~10-fold lower concentrations. These data suggest that endogenous PAM exerts an inhibitory influence on AC activity and that the decrease in endogenous PAM enhances basal AC activity and facilitates its activation by G protein-coupled receptors.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Antisense ODNs against PAM decrease PAM protein in HeLa cells, increase basal cAMP levels, and enhance the accumulation of cAMP in response to VIP. Panel A, HeLa cells were transfected with 3 µM antisense, sense, and antisense ODN harboring three-point mutations (3M-as) as described under "Materials and Methods." Cells were lysed in Laemmli sample buffer and subjected to Western analyses with anti-PAM antibody and anti-epidermal growth factor receptor antibody (EGFR). Panel B, lysates of HeLa cells that had or had not been transfected with sense, antisense, and 3M-as ODNs were assayed for AC activity in the presence of 100 µM forskolin. The means ± S.E. of three determinations are shown. Panel C, HeLa cells were treated with sense, antisense, and 3M-as ODNs as described under "Materials and Methods." Then 100 µM isobutylmethylxanthine was added for 5 min. The medium was aspirated; after washing the cells with PBS, 1 N HCl was added, and cells were frozen on dry ice. The cAMP measurements were performed by radioimmunoassay as described under "Materials and Methods." The means ± S.E. of three experiments are presented; *p  0.001; **p  0.01, Student's unpaired t test. Panel D, HeLa cells that had been transfected with sense or antisense ODNs as described above were treated for 5 min in the presence of 100 µM isobutylmethylxanthine followed by various concentrations of VIP for 10 min. After removing the medium, the reactions were terminated by the addition of 1 N HCl. Cyclic AMP accumulation in cells was determined as described under "Materials and Methods." Data from at least two experiments performed four times each are presented as the mean ± S.E.; * p  0.001 Student's unpaired t test.

Guo et al. (30) have previously shown PAM to be a nuclear protein during interphase and to be distributed throughout the cell during mitosis. Because AC is a membrane-bound protein, we investigated the cellular localization of PAM in HeLa cells using the antibody that Guo et al. (30) had developed. Our data (Fig. 6) demonstrate that during the M phase, PAM is located exclusively in the cytosol. On the other hand, during the G₁ phase, PAM is distributed both within the nucleus and the cytoplasm. The precise reason(s) for the slight differences in distribution of PAM in HeLa cells (Fig. 6) versus in endothelial cells (30) despite using the same antibody is presently not clear. It could be related to the different cell types. Nevertheless, the finding that PAM is located in cytosol during the M and G₁ phases (Fig. 6) coupled with the data that show that a decrease in PAM expression increases basal and agonist-stimulated cAMP accumulation (Fig. 5) indicate that PAM can modulate AC activity in intact cells.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   PAM changes its cellular location during the cell cycle. HeLa cells (1 × 105 cells/35-mm plate) were arrested with nocodazole for 24 h and then released from this block for 2 h (panels A-C) and 8 h (panels D-F) as described under "Materials and Methods." The cells were then fixed and stained with propidium iodide (red; panels B and E) to visualize the nuclei and anti-PAM antibody (green; panels A and D) as described under "Materials and Methods." Panels C and F represent merged images to monitor the localization of PAM in nuclei. In parallel experiments, the cell cycle phases were determined by fluorescence-activated cell sorter analyses.

PAM is a large protein that is associated with Myc (30). The region of Myc which associates with PAM is necessary for transcriptional activation by Myc, and mutations in this region have been observed in Burkitt's and AIDS-associated lymphomas (30). Whether or not this interaction of PAM with Myc has functional significance in these diseases remains unknown. More recently, homologs of PAM in C. elegans (RPM-1) and in Drosophila (HIW) have been shown to be important for synaptogenesis at the neuromuscular junctions (31-33). Thus, the mammalian PAM may play a similar role. However, because PAM is a large protein with a number of potentially important domains for biological action, it is likely that this protein has a number of biological functions. This notion is underscored by the fact that PAM is also expressed in a large variety of non-neuronal tissues and cells (27). In this respect, our studies have assigned mammalian PAM with a functional role as the most potent inhibitor of some isoforms of ACs. Clearly, the decrease in its intracellular concentration increases basal cAMP levels and also responsiveness of AC to agonists of seven-transmembrane receptors such as VIP, suggesting that endogenous PAM in mammalian cells tonically inhibits the AC signaling system. Although PAM clearly modulates the ability of G_s to stimulate AC activity, whether or not PAM also affects regulation of AC isoforms by G or other modulators such as Ca²⁺/calmodulin remains to be determined by future studies.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Q. Guo, NIH, Bethesda, MD for the clones corresponding to PAM cDNA and the anti-PAM antibody. We thank Dr. A. G. Gilman, University of Texas Southwestern Medical School for the G_s cDNA. We also thank Dr. R. Iyengar, Mt. Sinai Medical School for the baculovirus to express ACI and ACII in Sf9 cells.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL59679 (to T. B. P.) and by a grant from the American Heart Association, Southeast Consortium (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Klinische Pharmakologie, Klinikum der Universität Frankfurt, Theodor Stern Kai 7, D-60590 Frankfurt, Germany.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Tennessee Health Science Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-6006; Fax: 901-448-4828; E-mail: tpatel@physio1.utmem.edu.

Published, JBC Papers in Press, October 4, 2001, DOI 10.1074/jbc.M107816200

	ABBREVIATIONS

The abbreviations used are: AC, adenylyl cyclase; G_s, subunit of the stimulatory G protein of adenylyl cyclase, G_s*, constitutively active (Q213L) mutant of G_s; G_i, subunit of the inhibitory G protein G_i; G, subunits of heterotrimeric G proteins; PAM, protein associated with Myc; RCC1, regulator of chromosome condensation; DMEM, Dulbecco's modified Eagle's medium; GTPS, guanosine 5'-3-O-(thio) triphosphate; PBS, phosphate-buffered saline; ODN, oligodeoxynucleotide; VIP, vasoactive intestinal peptide.

	REFERENCES

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