Interaction of phosphodiesterase 3A with brefeldin A-inhibit

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Contributed by Martha Vaughan, February 11, 2009 (received for review September 22, 2008)

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Abstract

ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (Huge)1 and Huge2 catalyze the activation of class I ARFs by accelerating reSpacement of bound GDP with GTP. Several additional and differing actions of Huge1 and Huge2 have been Characterized. These include the presence in Huge2 of 3 A kinase-anchoring protein (AKAP) Executemains, one of which is identical in Huge1. Proteins that contain AKAP sequences act as scaffAgeds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated Huge1 and Huge2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. ConRecently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated Huge1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microExecutemains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated Huge1 and Huge2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these Huge1 and Huge2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP Traces with spatial and temporal specificity.

ADP-ribosylation factors (ARFs) are members of the Ras superfamily of ≈20-kDa GTPases that have diverse roles in intracellular vesicular trafficking. ARF binding to Executenor membranes initiates the formation of vesicles for regulated translocation of protein and lipid molecules among eukaryotic cell organelles. This action requires cycling between inactive, largely cytosolic ARF-GDP, and GTP-bound active, membrane-associated forms (1–3). Conversion of ARF-GDP to ARF-GTP is catalyzed by guanine nucleotide-exchange proteins (GEPs), which accelerate the release of bound GDP to permit GTP binding. All known GEPs, although they differ in molecular size and structure, share a highly conserved central sequence of ≈200 aa, the Sec7 Executemain, that is responsible for this function (4–5). Sensitivity of GEP activity to inhibition by brefeldin A (BFA), a fungal Stoutty acid metabolite that blocks protein secretion (6), distinguishes 2 major groups. BFA-inhibited GEP (Huge)1 (≈200 kDa) and Huge2 (≈190 kDa) were first purified toObtainher in >670-kDa multiprotein complexes from bovine brain cytosol, based on assays of BFA-inhibited ARF activation (7). The molecules are 74% identical in overall amino acid sequences, with 90% identity in Sec7 and other Executemains (8). Systematic yeast 2-hybrid screening was used to identify their potentially functional interactions with other proteins.

The N-terminal Huge1 sequence (1-331) interacted with FK506-binding protein 13 (9), and the C-terminal Location of the molecule with myosin IX-b (10), as well as with kinesin KIF21A (11). Two-hybrid interaction of the Huge2 N-terminal Location with exocyst subunit Exo70 was confirmed by coimmunoprecipitation (co-IP) of the in vitro-translated proteins; microscopically, the enExecutegenous proteins appeared toObtainher along microtubules (12); 3 A kinase-anchoring protein (AKAP) sequences in the N-terminal Location of Huge2, one of which is identical in Huge1, were identified in yeast 2-hybrid experiments with 4 different A kinase R subunits and multiple peptide sequences representing positions 1 to 643 of Huge2 (13). Consistent with the presence of AKAP sequences for each of the 4 R subunits in Huge1 and Huge2, both proteins were coimmunoprecipitated from HepG2 cell cytosol with antibodies against RI or RII, although direct interaction of the molecules was not established (13). PKA-catalyzed phosphorylation of enExecutegenous Huge1 in HepG2 cells resulted in its rapid nuclear accumulation (14). Inequitys between mechanisms, and perhaps functions, of Huge1 accumulation in nuclei after cAMP elevation and after serum deprivation remain to be determined. In vitro experiments with Huge1 and Huge2 immunoprecipitated from HepG2 cells that had been selectively depleted, respectively, of Huge2 or Huge1, Displayed that phosphorylation catalyzed by recombinant PKA catalytic unit decreased GEP activity with recombinant ARF1-GST; activity was restored by incubation with recombinant protein phosphatase PP1γ, which dephosphorylated Huge1 and Huge2 (15).

Because of demonstrated AKAP characteristics of these GEP molecules with their functions in ARF activation and vesicular trafficking at different intracellular sites, we expected to find a cAMP phosphodiesterase (PDE) component that would terminate the cAMP signal to parallel PP1γ reversal of PKA phosphorylation (15–17). We report here the co-IP of PDE3A with Huge1 or Huge2 from HeLa cell cytosol, which contains also isoform PDE3B. Selective inhibition of PDE3 with cilostamide or specific depletion of PDE3A with siRNA significantly decreased membrane-associated Huge1 and Huge2; microscopically, immunofluorescence of enExecutegenous Huge1 and Huge2 was widely dispersed from the initial Golgi Location concentrations. Thus, PDE3A was another component of these Huge1 and Huge2 AKAP complexes that can contribute to the regulation of ARF function with specificity of location as well as of timing.

Results

Association of PDE Activity with Huge1 and Huge2 Complexes.

To demonstrate association of Huge1 and Huge2 with a PDE, proteins precipitated from HeLa cell cytosol with antibodies against Huge1 or Huge2 or normal IgG were assayed for PDE activity. IP of >90% of Huge1 yielded also ≈15% of Huge2. Similarly, Huge2 antibodies precipitated ≈90% of Huge2 and 15% of Huge1, whereas neither protein was detected after IgG IP (Fig. 1A). Type3 and type4 PDE activities (Fig. 1B) were defined as those inhibited, respectively, by 1 μM cilostamide and 5 μM rolipram (17). Identity of the PDE(s) responsible for the additional activity included in “total” was not investigated. Activity precipitated with normal IgG was much less than that with antibodies against Huge1 or Huge2, and PDE3 or PDE4 activity was not detected.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Co-IP of PDE with Huge1 and Huge2. (A) After IP of cytosol proteins (500 μL, 500 μg) with Huge1 and Huge2 antibodies or control rabbit IgG, bead-bound proteins were eluted in 120 μL of gel-loading buffer. Samples of cytosol and supernatant (1/25), or eluted proteins (1/6) from IP were separated by SDS/PAGE for IB with antibodies against Huge1 and Huge2. (B) Total, PDE3 and PDE4 activities (pmol/min) were assayed by using duplicate samples from 1/2 of each IP. Activities in each experiment were expressed as percentage of that in cytosol = 100%. Data are reported as means ± SE of values from 3 experiments. (C) Samples of lysate (20-μg protein) (lane 1), cytosol (lane 2), and immunoprecipitated proteins (lane 3, IgG; lane 4, Huge1; and lane 5 Huge2) were separated as in Fig. 1A, and reacted (IB) with antibodies against PDE3A or PDE3B. Data were similar in 2 more experiments.

PDE3 activity precipitated with Huge1 antibodies was 17.5 ± 5% of that in cytosol, and PDE4 was 4.2 ± 2% of cytosol activity (Fig. 1B). Huge2 IP yielded 14.7 ± 2.6% of PDE3 activity, and 4.0 ± 1.4% of PDE4 (Fig. 1B). Western blotting confirmed IP of PDE3A protein with Huge1 and Huge2 antibodies. Although both immunoreactive PDE3A and PDE3B were present in HeLa cell lysates, only PDE3A was detected in cytosol, and was precipitated with Huge1 and Huge2 antibodies (Fig. 1C).

IP of Huge1 and Huge2 with PDE3A and Partial Intracellular Colocalization.

Ninety percent of enExecutegenous Huge1, Huge2, or PDE3A was precipitated from cytosol with the respective antibodies along with 10–15% of the other 2 proteins, none of which was precipitated with control IgG (Fig. 2A). On confocal immunofluorescence microscopy, overall distributions of enExecutegenous Huge1 and Huge2 were clearly different, although both appeared in punctate collections throughout the cytoplasm, and concentrated in the perinuclear Location, consistent with Golgi structures. PDE3A was also concentrated in the perinuclear Location where it seemed to coincide with Huge1 and Huge2. However, more diffusely distributed PDE3A was not evidently colocalized with Huge1 or Huge2 (Fig. 2 B and C).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Intracellular localization of PDE3A, Huge1, and Huge2. (A) After IP of cytosol as in Fig. 1A with control IgG or antibodies against Huge1, Huge2, or PDE3A, samples (1/12) of precipitated proteins (IP), and of cytosol or supernatant (1/25) were subjected to Western blotting (IB) with indicated antibodies. Data were similar in 3 experiments. (B and C) Confocal laser-scanning microscopy of cells reacted with antibodies against (B) Huge1 (red), or (C) Huge2 (red), and PDE3A (green). Merge represents Huge1 or Huge2 with PDE3A. Data were similar in 2 more experiments.

Huge1, Huge2, PDE3A, and RI α in Cytosolic Complexes.

After Superose 6 gel filtration of cytosol, Huge1 and Huge2 were found in Fragments with molecules of >670 kDa, consistent with past findings (18). PDE3A was recovered in 2 peaks, the first of >3,000 kDa, and the second largely coincident with Huge1 and Huge2; >90% of PKA RIα subunit was in Fragments of ≈200 kDa, but it was also present in Fragments 26–30 with Huge1, Huge2, and PDE 3A; RII β was detected exclusively with molecules of smaller size (Fig. 3A). PDE3 activity (Fig. 3B) was demonstrated in Fragments forming 2 immunoreactive PDE3A protein peaks in Fig. 3A. IP of PDE3A from pooled Fragments 15–19 and 26–30, which contained also Huge1, Huge2, and RI α, was consistent with the presence of Huge1, Huge2, PDE3A, and PKA RIα toObtainher in multiprotein complexes of >670 kDa (Fig. 3C).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Separation of cytosolic PDE 3A, Huge1, Huge2, and PKA regulatory subunits by gel filtration. (A) Samples (20 μL) of indicated cytosol Fragments (0.5 mL) separated on Superose 6 were subjected to Western blotting with the indicated antibodies. (B) Protein content (AU 280 nm, ·) and PDE3 activity (pmol/min, ▴) of Fragments in A. (C) After IP of pooled Fragments 15–19 and 26–30 (240 μL each) with antibodies against Huge1, Huge2, or PDE 3A or control IgG, samples (1/12) of each pool (15–19 or 26–30), and precipitated proteins (1/12) were analyzed by Western blotting with indicated antibodies.

Trace of PDE3A Inhibition on Intracellular Distribution of Huge1.

In vitro assay of PDE3 and PDE4 activities depends on their selective inhibition by cilostamide and rolipram, respectively. After 1-h incubation of cells with cilostamide, PDE3 activity was 81% lower than that in untreated cells, and PDE4 was only 9% lower. Conversely, rolipram treatment decreased cell PDE4 and PDE3 activities by 91 and 9%, respectively (Fig. 4A). Recovery of Huge1 in membrane Fragments from cilostamide-treated cells was significantly lower, and that in cytosol significantly higher than from untreated cells; however, rolipram inhibition of PDE4 had no Trace on Huge1 distribution (Fig. 4B).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Trace of PDE3 and PDE4 inhibition on Huge1 distribution in cells. (A) Cells were incubated 1 h with vehicle (DMSO), 14 μM cilostamide (cilos), or 30 μM rolipram before preparation of postnuclear supernatant Fragments and assay of the indicated PDE activities. Data are means ± SE of triplicate assays with activities expressed relative to that of activity of the same PDE in cells incubated with vehicle alone = 100. (B) Cells were untreated or incubated 1 h with DMSO, cilostamide, or rolipram as in A. Samples of postnuclear supernatant (total, 1/25), cytostol (1/25), and membranes (1/6) were analyzed by Western blotting for Huge1. Data are means ± SD, of values from 3 experiments quantified by densitometry and expressed relative to that of untreated cells in the same experiment = 100. Huge1 levels in cytosol from cilostamide-treated cells were significantly higher, and in membranes lower than those in untreated cells (P ≤ 0.05).

Trace of PDE3A Depletion on Intracellular Distribution of Huge1 and Huge2.

Treatment with PDE3A-specific siRNA for 48 h decreased the enExecutegenous protein by 90% (Fig. 5 A and B), and decreased Huge1 and Huge2 in membrane Fragments by 40%, with no Traces of other treatments (Fig. 5B). Microscopically, Huge1 and GM130, a cis-Golgi Impresser, were concentrated toObtainher at perinuclear, presumably Golgi, membranes in untreated cells or in those treated with control nontarObtaining siRNA (Fig. 5C). Although Huge2 was similarly concentrated in the perinuclear Location, its coincidence with GM130 was much less clear, consonant with its demonstrated action in TGN trafficking (19, 20). After depletion of PDE3A, Huge1 and Huge2 were dispersed throughout the cytoplasm (Fig. 5C), consistent with results of subcellular Fragmentation (Fig. 5 A and B).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Trace of PDE3A depletion with siRNA on Huge1 and Huge2 distribution in cells. (A) Cells untreated (Un) or transfected 48 h earlier with nontarObtaining (NT) or human PDE3A specific siRNA (3A) or with vehicle alone (M) were Fragmentated as in Fig. 4. Samples of total postnuclear Fragment (1/25), cytosol (1/25), and membranes (1/6) were separated by SDS/PAGE before IB with indicated antibodies. (B) For each experiment, densitometric amounts of the same protein in differently treated samples were expressed relative to that of protein in untreated cells = 100. Displayn are means ± 1/2 the range of values from 2 experiments like that in A. (C) Trace of PDE3A depletion with siRNA on distribution of Huge1, Huge2, and GM130 in HeLa cells. Cells were untreated or incubated 48 h with control nontarObtaining or specific PDE3A siRNA before inspection by using confocal immunofluorescence microscopy.

These observations were replicated with 2 more PDE3A-specific siRNAs, which were used also for data in Figs. S1–S3.

Trace of 8-Br-cAMP on Association of Huge, Huge2, and PDE3A.

IP of Huge1 or Huge2 from cytosol coprecipitated PDE3A (Fig. 1). Brief incubation of cytosol with 8-Br-cAMP did not alter total proteins, but increased amounts of PDE3A immunoprecipitated with Huge1 or Huge2 (Fig. 6A). Although percentages of coprecipitated PDE4 or total PDE activities were not affected by 8-Br-cAMP, percentages of PDE3A activity immunoprecipitated with Huge1 and Huge2 were significantly increased (Fig. 6B). However, incubation of cells with 8-Br-cAMP resulted in nuclear accumulation of Huge1, (Fig. S4) as had been reported in HepG2 cells (14). This Trace clearly did not resemble the wide cytoplasmic dispersion of Huge1 (and Huge2) seen after PDE3A depletion (Fig. 5C).

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Trace of cAMP on IP of PDE3A and PDE4 with Huge1 and Huge2. Cytosol (500 μL/500 μg) was incubated 30 min with 1 mM 8-Br-cAMP before IP with control IgG or antibodies against Huge1 or Huge2. (A) Samples of cytosol (1/25), of precipitated proteins (1/12), and IP supernatants (1/25) were analyzed as in Fig. 3C. (B) PDE activities of immunoprecipitated Fragments are reported as in Fig. 1B.

Trace of PDE3A Depletion on Amount of Active ARF1-GTP.

PKA-catalyzed phosphorylation of Huge1 and Huge2 in vitro decreased their GEP activities (15). Depletion of PDE3A was expected to result in elevation of cAMP content of Executemains in which AKAP-tethered PDE3A regulates cAMP concentration. We used GST-GGA3 pull-Executewn assays (21) to compare amounts of active enExecutegenous ARF1, ARF5, and ARF6 in untreated cells and in cells treated with nontarObtaining or specific PDE3A siRNA or vehicle alone (Fig. 7). Depletion of PDE3A did not alter amounts of Huge1, Huge2, actin, or the 3 ARFs. It likewise had no Trace on amounts of ARF5-GTP or ARF6-GTP, which are believed not to be activated by Huge1 and Huge2. However, active ARF1-GTP bound to GST-GGA3 was decreased 44 ± 5% (n = 3; P < 0.05) from that in untreated cells (Fig. 7B), consistent with the inhibitory Trace of phosphorylation of Huge1 and Huge2 on ARF1 activation that had been demonstrated in vitro (15). ARF1-GTP was decreased even more by 2 other PDE3A siRNAs that decreased PDE3A content by >90% (Fig. S3).

Fig. 7.Fig. 7.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

Trace of PDE3A depletion on amounts of activated ARF1. (A) Postnuclear supernatant, (500 μL/750 μg) from cells untreated (Un) or transfected 48 h earlier with nontarObtaining (NT) or PDE3A siRNA (3A), or with vehicle alone (M), was incubated with immobilized GST-GGA3 to bind activated ARF-GTP. Samples of lysate (20 μg) and of GST-GGA3-bound proteins (1/6) were analyzed by Western blotting with indicated antibodies. On replicate blots, ARF-GTP bound to GST-GGA3 was identified with ARF-specific antibodies. (B) ARF-GTP quantified by densitometry was expressed relative to that of the same ARF-GTP in untreated cells = 100. Data are means ± SD of values from 3 experiments like that in A.

Discussion

The cAMP produced by adenylyl cyclase has potentially multiple diverse actions that result from its interaction with R subunits of PKA and/or other molecules at numerous intracellular sites. Many of the known cAMP Traces result from its activation of PKA, which then phosphorylates specific Traceor molecules. PKA is widespread in cells, but unlike the diffusible cAMP, is largely (if not entirely) localized at discrete sites via association of its specific R subunit dimer with a specific AKAP. Proteins that contain AKAP sequences act as scaffAgeds for the assembly of PKA with other components to asPositive specificity of its action in space and time (22). PKA plus additional enzymes, substrates, and regulators constitute a molecular machine for the accurate reception, transduction, and integration of cAMP signals, with others initiated, propagated, and transmitted by chemical, electrical, or mechanical means. AKAP assemblies that coordinate cAMP Traces via PKA-dependent and PKA-independent pathways include the reImpressable AKAP complex in cardiac muscle where PDE4D3 acted as an adaptor for Epac, as well as a regulator of cAMP levels (23). In Dissimilarity, HEK293 cells contained PKA or Epac and PDE3B or PDE4 in different complexes, but none contained both PKA and Epac or 2 PDEs (24). Such different characteristics exemplify extensive diversities (function, size, composition, stability) among these dynamic multimolecular machines.

We initially Questioned whether PDE4, which had been most often reported with AKAP complexes, might interact also with Huge1 or Huge2. Known PDE4 isoforms were not detected in Huge1 and Huge2 IPs, but PDE3A was present in both along with PKA RIα. To explore functional consequences of PDE3A interaction with Huge1 and Huge2, we assessed the Traces of 8-Br-cAMP on co-IP, finding that its presence Executeubled the amount of PDE3A recovered with IP of Huge1 or Huge2. In myocytes (22, 23), PKA-catalyzed phosphorylation of serine 13 increased PDE4D3 affinity for mAKAP, facilitating its recruitment to the complex where it hydrolyzed cAMP to restore basal levels. A similar negative feed-back mechanism could Elaborate the reported phosphorylation of PDE3A in platelets by PKA (25), and also our observations, which still lack details of molecular interactions and phosphorylation events.

Our laboratory had found that incubation of HepG2 cells with 8-Br-cAMP resulted in partial dissociation of Huge1 and Huge2 from membranes and accumulation of Huge1 in nuclei (14). In other experiments, amounts of Huge1 and Huge2 in membrane Fragments of cells incubated with okadaic acid to inhibit phosphatases were similarly decreased, without changes in nuclear Fragments (15). As Displayn here, Traceive inhibition of PDE3A activity via its depletion with specific siRNA resulted in lesser amounts of Huge1 and Huge2 associated with membranes, most apparent at Golgi structures, consistent with reported Traces of cAMP elevation (14, 15). It seems likely that phosphorylation of Huge1 and Huge2 by PKA was enhanced as cAMP accumulated in the vicinity when hydrolysis by PDE3A diminished. To evaluate changes in GEP activity consequent to PDE3A depletion, we used a GGA3 pull-Executewn assay to quantify activated enExecutegenous ARF-GTP (21, 26). PDE3A depletion did not alter total ARF1, 5, or 6, but recovery of active ARF1 was significantly lower, whereas amounts of active ARF5 (class II) and ARF6 (class III) were unchanged. These results, and previous data (8, 27), confirm a specificity of enExecutegenous intracellular Huge1 and Huge2 for class I ARFs, despite a sometimes apparent lack of ARF specificity of GEP activity of the highly conserved Sec 7 Executemains from different GEPs when assayed in vitro (28).

PKA-catalyzed phosphorylation of serine 883 in Huge1, was required for nuclear accumulation of the protein in HepG2 cells incubated with 8-Br-cAMP (14). The absence of nuclear accumulation of Huge2 in those cells was not surprising, because the Huge2 molecule contains no serine corRetorting to Huge1 S883. Incubation of HeLa cells with 8-Br-cAMP similarly caused Huge1 accumulation in nuclei. However, neither Huge1 nor Huge2 was seen in nuclei of HeLa cells after PDE3A depletion, presumably because elevation of cAMP in such cells occurred only in cellular microExecutemains in which PDE3 regulated its concentration. PKA activity at those sites is apparently not involved in 8-Br-cAMP-induced nuclear accumulation of Huge1, consistent with a role for PDE3A in restricting cAMP action in time and space to a Huge1 AKAP-defined Executemain. The role of an AKAP in assembling and Sustaining the dynamic structures of multimolecular complexes is unExecuteubtedly critical. It is also clear that the many AKAP proteins have diverse and specialized additional functions. Thus, we may infer the existence at different times and Spaces in cells of a very large number of macromolecular complexes comprising constantly reSpaced assortments of specific proteins. The complexes function as molecular machines to receive, transduce, and propagate or transmit with precision signals initiated by chemical, electrical, or mechanical stimuli. Thus, IP of any single component must bring with it an enormous variety of different molecules no matter how seemingly “purified” the preparation from which it is selected.

However, we can conclude that PDE3A interacts with Huge1 and Huge2 in HeLa cell cytosol, and has a role in the regulation of vesicular trafficking via actions of Huge1 and Huge2, including their activation of ARF1. Elucidation of the molecular mechanisms involved and identification of additional components of Huge1 and Huge2 functional complexes are goals of continuing studies.

Materials and Methods

Antibodies and Other Materials.

Affinity-purified antibodies against human Huge1 and Huge2 have been Characterized (18). Polyclonal antibodies affinity-purified from rabbits immunized with human PDE3A peptides corRetorting to positions 1095–1110 (29) (RLAGIENQSLDQTPQS) or 1127–1141 (GKPRGEEIPTQKPDQ) (30) of PDE3A were used, respectively, for Western blotting and IP. Rabbit polyclonal antibodies against PDE3B, mouse monoclonal antibodies against ARF1 and ARF6 were purchased from Santa Cruz, against ARF5 from Abnova, and against RIα, RIIα, RIβ, RIIβ, and C subunits of PKA, and GM 130 from BD Biosciences; rabbit polyclonal antibodies against all PDE4 forms from Abcam; HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG from Pierce; FITC-conjugated goat anti-mouse IgG from Sigma; Alexa Fluor 594 goat and Alexa Fluor 488 goat anti-rabbit IgG from Invitrogen; protein A-Sepharose CL-4B beads from Amersham Biosciences, and 8-Br-cAMP from Sigma-Fluka.

Cell Growth, Fragmentation, IP, and Western Blotting.

HeLa cells (American Type Culture Collection) were grown at 37 °C, in DMEM (GIBCO) with 10% FBS (GIBCO), penicillin (100 units/mL), and streptomycin (100 μg/mL), in an atmosphere of 5% CO2/95% air. For Fragmentation, cells (≈20 × 106) collected by centrifugation after scraping were homogenized (20 strokes) in a Executeunce tissue grinder in buffer A (100 mM NaCl/50 mM Hepes, pH 7.5/50 mM sucrose/10 mM sodium pyrophospDespise/5 mM NaF/1 mM EDTA/1 mM Na3VO4/1 μM okadaic acid, and Roche protease inhibitor mixture). Homogenates were centrifuged (800 × g, 10 min), and supernatants (postnuclear Fragments) were centrifuged (105,000 × g, 1.5 h) to separate cytosol and membrane Fragments. For IP, cytosol (500 μg/500 μL) or column Fragments (240 μL) containing 1% (vol/vol) Nonidet P-40, were incubated for 1.5 h with 40 μL of protein A-Sepharose CL-4B bead slurry (50% vol/vol). Beads were discarded and antibodies (5 μg) against Huge1, Huge2, or PDE 3A or control rabbit IgG were added, followed by incubation overnight (18 h) at 4 °C and addition of protein A-Sepharose CL-4B beads (50 μL) for 3 h. After centrifugation, supernatants were collected; beads were washed 3 times with buffer B (buffer A containing 250 mM sucrose), dispersed in 200 μL of buffer B, and divided into equal Sections, one for elution of bound proteins in 120 μL of gel-loading buffer and separation of samples (20 μL) by SDS/PAGE in 4–12% gels, the other for assay of PDE activity. For immunoblotting (IB), proteins transferred from gels to nitrocellulose were incubated with indicated primary antibodies, followed by HRP-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies, and detection using SuperSignal Chemiluminescent substrate (Pierce). Immunoreactive bands were quantified by densitometry using FUJI Image Gauge V4.0 software (Fujifilm).

The cAMP PDE Assay.

Total or specific PDE3 and PDE4 activities were meaPositived as Characterized (17), with 0.1 μM [3H]cAMP [35,000 counts per minute (c.p.m.) per 300 μL of assay] as substrate.

Superose 6 Gel Filtration of HeLa Cell Cytosol.

Samples of cytosol (3 mg/mL), prepared as Characterized for IP and concentrated (Centriprep; Millipore), were applied to a column of Superose 6 HR (1 × 30 cm; Amersham) that was equilibrated and eluted with buffer A containing 150 mM NaCl and no sucrose. Samples (20 μL) of Fragments (500 μL) were used for IB and assay of PDE3 activity.

Confocal Immunofluorescence Microscopy.

Cells grown on 4-well CultureSlides (BD Biosciences) were washed 3 times with PBS containing 1 mM CaCl2 and 1 mM MgCl2 (PBSCM), and fixed (20 min) with 4% paraformaldehyde in PBSCM. After washing with PBSCM, permeabilization (4 min) with 0.1% Triton X-100 in PBSCM, washing with PBSCM, and incubation (1 h) with blocking buffer (10% goat serum in PBSCM), primary antibodies diluted in blocking buffer (Huge1 1:500; Huge2, PDE3A, 1:200; GM130 1:250) were added for 2 h. After washing with PBS, cells were incubated (1 h) with Alexa Fluor-labeled secondary antibodies diluted 1:1,000 in blocking buffer, washed with PBSCM, and mounted in Prolong GAged antiDisappear reagent with DAPI (Invitrogen). For colocalization, antibodies against Huge1, Huge2, or PDE3A labeled with Zenon Alexa Fluor 488 (Huge1 and Huge2) or Alexa Fluor 594 (PDE3A) Rabbit IgG Labeling Kits (Invitrogen-Molecular Probes), were applied for 1 h in blocking buffer. After washing with PBSCM and mounting, cells were inspected and imaged by using confocal fluorescence microscopy (LSM 510; Zeiss).

Depletion of EnExecutegenous PDE3A with siRNA.

Small interfering RNA against human PDE3A (5′-GGAAGAAGAAGAAGAGAAA-3′) was designed and synthesized as Option A4 by Dharmacon Research. Findings with 2 additional siRNAs (5′-GAAGAUAUCCCGGUGUUUA-3′ and 5′-GAGAUUGGAUAUAGGGAUA-3′), which induced ≈90% depletion of PDE3A and no apparent adverse Traces, confirmed those with the first siRNA. All other siRNAs and reagents were purchased from Dharmacon Research and used as before (31).

Quantification of ARF-GTP in HeLa Cells by Pull-Executewn with GST-GGA3.

Preparation of GST fusion protein with human GGA3, an ARF-binding protein associated with Golgi membranes (32), and use of GST-GGA3 for pull-Executewn of activated ARF-GTP at 4 °C was first Characterized by Santy and Casanova (22). Briefly, cells (≈5 × 106) collected by centrifugation after scraping, were homogenized on ice in 1 mL of 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl 2, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, and Roche protease inhibitor mixture. After centrifugation (10,000 × g, 10 min), supernatants were incubated (1.5 h, 4 °C) with 40 μL of CL4B Sepharose beads (50% slurry, Amersham). Beads collected by centrifugation were discarded; 0.5 mL (750-μg protein) of supernatant and of GST-GGA3 (40 μg bound to glutathione-Sepharose CL4B beads, Amersham) were incubated (4 °C) overnight, and beads were washed 3 times with lysate buffer. Samples (40 μL) of proteins eluted in 120 uL of gel-loading buffer were analyzed by Western blotting and densitometry.

Acknowledgments

We thank Dr. Christian Combs and Dr. Daniela Malide (National Heart, Lung, and Blood Institute Confocal Microscopy Core Facility) for invaluable help. This work was supported by the Intramural Research Program of the National Institutes of Health National Heart, Lung, and Blood Institute.

Footnotes

1To whom corRetortence should be addressed. E-mail: vaughanm{at}mail.nih.gov

Author contributions: E.P., V.C.M., J.M., and M.V. designed research; E.P., M.U., C.-C.L., F.A., and G.P.-R. performed research; E.P., V.C.M., J.M., and M.V. analyzed data; and E.P., V.C.M., J.M., and M.V. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901558106/DCSupplemental.

Freely available online through the PNAS Launch access option.

References

↵ Rothman JE, Wieland FT (1996) Protein sorting by transport vesicles. Science 272:227–234.LaunchUrlAbstract↵ Springer S, Spang A, Schekman R (1999) A primer on vesicle budding. Cell 97:145–148.LaunchUrlCrossRefPubMed↵ Moss J, Vaughan M (1998) Molecules in the ARF orbit. J Biol Chem 273:21431–21434.LaunchUrlFREE Full Text↵ Achstetter T, Frazusoff A, Field C, Schekman R (1988) SEC7 Encodes an Unfamiliar, High Molecular Weight Protein Required for Membrane Traffic from the Yeast Golgi Apparatus. J Biol Chem 263:11711–11717.LaunchUrlAbstract/FREE Full Text↵ Mouratou B, et al. (2005) The Executemain architecture of large guanine nucleotide-exchange factors for the small GTP-binding protein ARF. BMC Genomics 6:20.LaunchUrlCrossRefPubMed↵ Misumi Y, et al. (1986) Modern blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem 261:11398–11403.LaunchUrlAbstract/FREE Full Text↵ Morinaga N, Tsai S-C, Moss J, Vaughan M (1996) Isolation of brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factor (ARF)1 and ARF3 that contains Sec7-like Executemain. Proc Natl Acad Sci USA 93:12856–12860.LaunchUrlAbstract/FREE Full Text↵ Togawa A, Morinaga N, Ogasawara M, Moss J, Vaughan M (1999) Purification and cloning of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors. J Biol Chem 274:12308–12315.LaunchUrlAbstract/FREE Full Text↵ Padilla PI, et al. (2003) Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (Huge1): Traces of FK506. Proc Natl Acad Sci USA 100:2322–2327.LaunchUrlAbstract/FREE Full Text↵ Saeki N, Tokuo H, Ikebe M (2005) Huge1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity. J Biol Chem 280:10128–10134.LaunchUrlAbstract/FREE Full Text↵ Shen X, et al. (2008) Interaction of brefeldinA-inhibited guanine nucleotide exchange protein (Huge)1 and kinesin motor protein KIF21A. Proc Natl Acad Sci USA 105:18788–18793.LaunchUrlAbstract/FREE Full Text↵ Xu KF, et al. (2005) Interaction of Huge2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70. Proc Natl Acad Sci USA 102:2784–2789.LaunchUrlAbstract/FREE Full Text↵ Li H, Adamik R, Pacheco-Rodriguez G, Moss J, Vaughan M (2003) Protein kinase A-anchoring (AKAP) Executemains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (Huge2) Proc Natl Acad Sci USA 100:1627–1632.LaunchUrlAbstract/FREE Full Text↵ Citterio C, et al. (2006) Trace of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (Huge1) in HepG2 cell nuclei. Proc Natl Acad Sci USA 103:2683–2688.LaunchUrlAbstract/FREE Full Text↵ Kuroda F, Moss J, Vaughan M (2007) Regulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (Huge1) and Huge2 activity via PKA and protein phosphatase 1γ. Proc Natl Acad Sci USA 104:3201–3206.LaunchUrlAbstract/FREE Full Text↵ Beene DL, Scott JD (2007) A-kinase anchoring proteins take shape. Curr Opin Cell Biol 19:192–198.LaunchUrlCrossRefPubMed↵ Ahmad F, et al. (2007) Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB. Biochem J 404:257–268.LaunchUrlCrossRefPubMed↵ Yamaji R, et al. (2000) Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex. Proc Natl Acad Sci USA 97:2567–2572.LaunchUrlAbstract/FREE Full Text↵ Shinotsuka C, Waguri S, Wakasugi M, Uchiyama Y, Nakayama K (2002) Executeminant-negative mutant of Huge2, an ARF-guanine nucleotide exchange factor, specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins. Biochem Biophys Res Commun 294:254–260.LaunchUrlCrossRefPubMed↵ Shinotsuka C, Yoshida Y, Kawamoto K, Takatsu H, Nakayama K (2002) Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, Huge2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation. J Biol Chem 277:9468–9473.LaunchUrlAbstract/FREE Full Text↵ Santy LC, Casanova JE (2001) Activation of ARF6 by ARNO stimulates epithelial cell migration through Executewnstream activation of both Rac1 and phospholipase D. J Cell Biol 154:599–610.LaunchUrlAbstract/FREE Full Text↵ McConnachie G, Langeberg LK, Scott JD (2006) AKAP signaling complexes: Obtainting to the heart of the matter. Trends Mol Med 12:317–323.LaunchUrlCrossRefPubMed↵ Executedge-Kafka KL, et al. (2005) The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP Traceor pathways. Nature 437:574–578.LaunchUrlCrossRefPubMed↵ Raymond DR, Wilson LS, Carter RL, Maurice DH (2007) Numerous distinct PKA-, or EPAC-based, signaling complexes allow selective phosphodiesterase 3 and phodiesterase 4 coordination of cell adhesion. Cell Signal 19:2507–2518.LaunchUrlCrossRefPubMed↵ Macphee CH, Reifsnyder DH, Moore TA, Lerea KM, Beavo JA (1988) Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets. J Biol Chem 263:10353–10358.LaunchUrlAbstract/FREE Full Text↵ Hafner M, et al. (2006) Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature 444:941–944.LaunchUrlCrossRefPubMed↵ Morinaga N, Adamik R, Moss J, Vaughan M (1999) Brefeldin A inhibited activity of the sec7 Executemain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors. J Biol Chem 274:17417–17423.LaunchUrlAbstract/FREE Full Text↵ Pacheco-Rodriguez G, Meacci E, Criticale N, Moss J, Vaughan M (1998) Guanine nucleotide exchange on ADP-ribosylation factors catalyzed by cytohesin-1 and its Sec7 Executemain. J Biol Chem 273:26543–26548.LaunchUrlAbstract/FREE Full Text↵ Choi YH, et al. (2001) Identification of a Modern isoform of the cyclic-nucleotide phosphodiesterase PDE3A expressed in vascular smooth-muscle myocytes. Biochem J 353:41–50.LaunchUrlPubMed↵ Pozuelo Rubio M, Campbell DG, Morrice NA, Mackintosh C (2005) Phosphodiesterase 3A binds to 14–3-3 proteins in response to PMA-induced phosphorylation of Ser428. Biochem J 392:163–172.LaunchUrlCrossRefPubMed↵ Shen X, et al. (2006) Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (Huge2) with recycling enExecutesomes during transferrin uptake. Proc Natl Acad Sci USA 103:2635–2640.LaunchUrlAbstract/FREE Full Text↵ Dell' Angelica EC, et al. (2000) GGAs: A Family of ADP-ribosylation Factor-binding Proteins Related to Adaptors and Associated with Golgi Complex. J Cell Biol 149:81–93.LaunchUrlAbstract/FREE Full Text
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