Identification of the Insulin-Regulated Interaction of Phosphodiesterase 3B With 14-3-3 β Protein

  1. Hiroshi Onuma1,
  2. Haruhiko Osawa1,
  3. Kazuya Yamada2,
  4. Takahiro Ogura1,
  5. Fumiko Tanabe1,
  6. Daryl K. Granner3 and
  7. Hideichi Makino1
  1. 1Department of Laboratory Medicine, Ehime University School of Medicine, Ehime, Japan
  2. 2Department of Biochemistry, Fukui Medical University and CREST, Japan Science and Technology, Fukui, Japan
  3. 3Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee

    Abstract

    Phosphodiesterase (PDE)-3B, a major PDE isoform in adipocytes, plays a pivotal role in the antilipolytic action of insulin. Insulin-induced phosphorylation and activation of PDE3B is phosphatidylinositol 3-kinase (PI3-K) and Akt dependent, but the precise mechanism of PDE3B activation is not fully understood. We have identified 14-3-3 β, a critical scaffolding molecule in signal transduction, as a protein that interacts with PDE3B using the yeast two-hybrid system. The interaction between PDE3B and 14-3-3 β was then confirmed in vitro. The glutathione S-transferase (GST)-tagged 14-3-3 β interacts with endogenous PDE3B of rat adipocytes, and this interaction is enhanced when adipocytes are treated with insulin. Coimmunoprecipitation experiments reveal that endogenous PDE3B also associates with endogenous 14-3-3 β in rat adipocytes, and this interaction is enhanced by insulin. Two different PI3-K inhibitors, wortmannin and Ly294002, block this induction, suggesting that PI3-K is required. Synthetic 15 amino acid peptides of rat PDE3B containing phosphorylated Ser-279 or -302 inhibit this interaction, indicating that the insulin-regulated phosphorylation of these serine residues is involved. Because insulin receptor substrate-1 also associates with 14-3-3, the dimeric 14-3-3 β could function as a scaffolding protein in the activation of PDE3B by insulin.

    Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP or cGMP, the second messengers in various biological processes, and thereby regulate the concentration of these molecules in cells. There are at least 11 members of the PDE family (1). PDE3B, a major isoform in adipocytes, plays a pivotal role in the antilipolytic action of insulin. Insulin-induced activation of PDE3B decreases intracellular cAMP levels, which results in the dephosphorylation and inactivation of hormone-sensitive lipase and the consequent inhibition of lipolysis (25).

    The phosphorylation and activation of PDE3B by insulin is phosphatidylinositol 3-kinase (PI3-K)-dependent and does not involve the p70 ribosomal protein S6 kinase (p70 S6 kinase) or mitogen-activated protein kinase signaling pathways (6). The phosphorylation site of PDE3B is controversial. Rahn et al. (7) reported that insulin promotes the phosphorylation of rat PDE3B on Ser-302, which corresponds to Ser-296 in mouse PDE3B. Kitamura et al. (8) reported that insulin results in the phosphorylation of mouse PDE3B on Ser-273 (rat Ser-279), which is also a target of phosphorylation by Akt. Recently, it was reported that activated Akt phosphorylates and activates PDE3B in vitro (9). Whereas Akt is one of several possible insulin-dependent PDE3B kinases, the precise mechanism of PDE3B activation remains unknown.

    The seven known mammalian isoforms of 14-3-3 compose a family of highly conserved proteins that are expressed in all eukaryotic cells. In addition, 14-3-3 proteins associate with a variety of proteins involved in signal transduction, cell cycle regulation, and apoptosis by recognizing a phosphoserine surrounded by specific sequences such as RSXpSXP (X is any amino acids and pS is phosphorylated serine) (10). Thus, 14-3-3 proteins seem to play an important role in signaling pathways mediated by serine/threonine protein kinases.

    Further, 14-3-3 proteins interact with Ras, Raf-1, and PI3-K, suggesting that, through serine phosphorylation, they mediate growth factor signaling at common upstream sites. Insulin receptor substrate-1 (IRS-1), which participates in insulin signaling by interacting with SH2-domain-containing proteins through phosphotyrosine residues, also associates with 14-3-3 proteins (11,12). Although a role of 14-3-3 proteins in specific actions of insulin has not been documented, we show here that 14-3-3 β is an interacting modulator of PDE3B, a key enzyme involved in the antilipolytic action of insulin.

    RESEARCH DESIGN AND METHODS

    Yeast two-hybrid screening.

    The cDNA encoding the regulatory region of mouse PDE3B (amino acid [aa] sequence between 223 and 636) (mPDE3B[aa 223–636]) was ligated, in frame, to the DNA-binding domain of GAL4 in a pGBT9 vector. The yeast strain PJ69-4A, which has HIS3, ADE2, and lacZ reporters, was transformed with mPDE3B (aa 223–636) in pGBT9 using the LiAc method (13). As the first screen, the PJ69-4A strain transformed with mPDE3B (aa 223–636) in the pGBT9 plasmid was used to screen the 3T3L1 adipocyte cDNA library in pGAD GH. Approximately 3.2 × 106 primary transformants were obtained from the 3T3L1 adipocyte cDNA library. These transformants were first selected on SD-(−)His(−)Trp(−)Leu plates containing 20 mmol/l 3-aminotriazole at 30°C for 7 days, and then the positive clones were selected on SD-(−)Ade(−)Trp(−)Leu plates using the ADE2 reporter of the PJ69-4A yeast strain to confirm the specificity of the positive clones. As another confirmation of protein-protein interactions involving mPDE3B, the lacZ reporter of the PJ69-4A yeast strain was examined in His+ Ade+ Trp+ Leu+ clones by measuring β-galactosidase (β-gal) activity using a filter assay. Plasmid DNA from positive yeast clones, which were both His+ Ade+ Trp+ Leu+ and β-gal activity positive, was transformed into the Escherichia coli (HB101) by electroporation. Positive plasmid clones derived from the 3T3L1 adipocyte cDNA library were isolated. As the second screen, PJ69-4A was transformed simultaneously with each isolated positive plasmid and mPDE3B (aa 223–636) in pGBT9. His+ Ade+ Trp+ Leu+ and β-gal activity positive clones were selected again, as described above, except that β-gal activity was measured using a liquid assay. The DNA sequence of each clone was determined using an ABI 310 instrument, and nucleotide sequences from each positive clone were compared with those entered in the GenBank database using the BLAST sequence search and comparison program.

    Glutathione S-transferase pull-down assay.

    The cDNA encoding FLAG-tagged full-length or truncated forms of mouse PDE3B was ligated, in frame, to pET 3a vector. The FLAG-tagged full-length or truncated forms of mouse PDE3B, represented as mPDE3B (aa 1–322), mPDE3B (aa 223–636), mPDE3B (aa 621–1,100), or mPDE3B (full-length), were labeled with [35S]methionine (Amersham Pharmacia, Buckinghamshire, U.K.) using the T7 TNT Quick-coupled Transcription/Translation System (Promega, Madison, WI). The BL21 bacterial strain transformed with pGEX 3X-14-3-3 β was grown in LB media, and expression of the chimeric proteins was induced with 0.4 mmol/l isopropyl-β-thiogalactoside at 37°C for 4 h. Chimeric proteins were affinity-purified using glutathione resin (Novagen, Darmstadt, Germany) according to the manufacturer’s protocol. The radiolabeled PDE3B proteins and either glutathione S-transferase (GST)·14-3-3 β- or GST-glutathione resin complex was resuspended in interaction buffer (10 mmol/l Tris-HCl [pH 8.0], 50 mmol/l NaCl, 5 mmol/l EDTA, 0.1% Triton X-100, 1 mmol/l phenylmethylsulfonyl fluoride (PMSF), 1 mmol/l DTT) and incubated for 30 min at room temperature with continuous gentle mixing. A sample representing 10% of the input-labeled proteins and the entire pull-down complexes were subjected to SDS-PAGE. The gel was dried, and 35S-labeled proteins were visualized using a BAS 3000 (Fuji, Tokyo, Japan) image analyzer.

    Preparation of solubilized membrane fraction (solubilized P-2) and cytosol fraction of rat adipocytes.

    Isolated adipocytes were prepared from epididymal fat tissue of Sprague-Dawley rats using the collagenase method. The cells were incubated with or without insulin (3 nmol/l) in Krebs Ringer Albumin HEPES buffer at 37°C for 10 min. In some experiments, adipocytes were preincubated with okadaic acid (1 μmol/l) or wortmannin (100 nmol/l) at 37°C for 20 or 30 min, respectively. The cells were then homogenized in buffer A (50 mmol/l Tris HCl [pH 7.5], 1 mmol/l EDTA, 1 mmol/l EGTA, 50 mmol/l NaF, 5 mmol/l sodium pyrophosphate, 10 mmol/l sodium β-glycerophosphate, 0.1 mmol/l PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 μmol/l microcystin, 0.5 mmol/l sodium orthovanadate) in a Dounce tissue grinder. After brief centrifugation of the homogenates, the supernatant without the fat cake was centrifuged (100,000g) for 1 h at 4°C, and the membrane (P-2) and cytosol fractions were obtained. P-2 was then solubilized with 1% triton X-100 in buffer A and centrifuged (100,000g) for 1 h at 4°C, and the supernatant (solubilized P-2) was obtained.

    In vitro association of endogenous PDE3B with GST·14-3-3 β.

    Solubilized P-2 (200 μg protein) from rat adipocytes was incubated with GST glutathione resin or GST·14-3-3 β glutathione resin at 4°C overnight with continuous mixing. The resin-bound complexes were collected by centrifugation, subjected to SDS-PAGE, and then electrotransferred to a polyvinylidene fluoride membrane. Immunoblotting was performed using an anti-PDE3B antibody raised against a synthetic rat PDE3B peptide (6). The amount of the added GST fusion proteins was assessed by amido black staining of the electrotransferred membrane.

    The association of endogenous PDE3B with endogenous 14-3-3 β in rat adipocytes.

    Solubilized P-2 (200 μg protein) obtained from rat adipocytes incubated with or without 3 nmol/l insulin at 37°C for 10 min was incubated with an anti-14-3-3 β antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight, followed by the addition of protein G-agarose for last 2 h. The agarose-bound immune complex was collected by centrifugation, and washed twice with each of 1% Triton X-100 in 50 mmol/l HEPES pH 7.5 and 0.1% Triton X-100 in 50 mmol/l HEPES at pH 7.5. The complexes were then subjected to SDS-PAGE. The amount of PDE3B in the immune complex was quantified by immunoblotting using an anti-PDE3B antibody. In the experiment of peptide inhibition, solubilized P-2 was incubated with the synthesized peptide (1 mmol/l) at 4°C overnight before immunoprecipitation. For peptide inhibition experiments, Ser-phosphorylated (pS279, pS302, and pS427) or nonphosphorylated (S279, S302, and S427) peptides, each of which consists of 15 amino acids corresponding to the sequence around Ser-279, Ser-302, and Ser-427 in rat PDE3B, respectively, were synthesized (Fujiya, Tokyo, Japan). The peptide sequences were PVIRPRRR[pS]SCVSLG(pS279), SGKMFRRP[pS]LPCISR(pS302), and TAQLRRS[pS]GASGLLT(pS427), and their nonphosphorylated serine-containing counterparts (S279, S302, and S427, respectively) had the same surrounding sequences.

    Assay of PDE activity.

    PDE activity was assayed as described previously (14). Briefly, samples were incubated with 0.1 μmol/l [3H]cAMP (Amersham Pharmacia) for 5 min at 30°C, and the formed [3H]AMP was degraded by 5′-nucleotidase of snake venom to [3H]adenosine. The latter was isolated by chromatography on AG 1-X2 (Bio-Rad, Hercules, CA), and the radioactivity was measured by liquid scintillation counter.

    RESULTS AND DISCUSSION

    Yeast two-hybrid screening.

    We used the yeast two-hybrid system for the detection of PDE3B interacting proteins. A 3T3L1 adipocyte cDNA library (15) was screened using mouse PDE3B as the bait. The cDNA encoding the regulatory region of mouse PDE3B (aa sequence between 223 and 636) (mPDE3B [aa 223–636]) was ligated, in frame, to the DNA-binding domain of GAL4 in a pGBT9 vector. Approximately 3.2 × 106 independent clones were screened to obtain 40 positive clones, 5 of which were identified as 14-3-3 isoforms by DNA sequencing after a second screening. One of these corresponded to isoform 14-3-3 β and was selected for further study.

    In vitro association between synthesized PDE3B and GST·14-3-3 β.

    The GST pull-down assay was used to confirm an in vitro interaction between mouse PDE3B and 14-3-3 β. The FLAG-tagged full-length or truncated forms of mouse PDE3B were 35S-labeled using an in vitro transcription/translation system. Each of the labeled PDE3B proteins was incubated with glutathione-resin bound GST·14-3-3 β. GST·14-3-3 β interacted with mPDE3B (aa 1–322), mPDE3B (aa 223–636), and mPDE3B (full-length) but not with mPDE3B (aa 621–1,100). GST alone did not interact with any of these proteins (Fig. 1). The region of mouse PDE3B that interacts with 14-3-3 β is thus located between aa 223 and 322. It is interesting that this region of mouse PDE3B contains the two reported insulin-dependent phosphorylation sites, namely mouse Ser-273 (rat Ser-279) (8) and rat Ser-302 (7).

    The association between PDE3B and 14-3-3 β is insulin-inducible.

    Cilostamide, an inhibitor of PDE3, was used to confirm that the PDE activity in the solubilized membrane fraction of rat adipocytes (solubilized P-2) reflects PDE3B activity. PDE activity was increased 2.5-fold compared with the basal activity by 3 nmol/l insulin (Fig. 2A). Approximately 90% of both basal and insulin-induced PDE activity was inhibited by 1 μmol/l cilostamide (Fig. 2A, hatched bars). Because PDE3B is known to be a major isoform of PDE3 in adipocytes, the PDE activity measured primarily reflects PDE3B activity.

    GST·14-3-3 β was used as the bait to examine whether this chimeric protein can interact with endogenous PDE3B in rat adipocytes and to determine whether this interaction is insulin-inducible. Glutathione resin-bound GST·14-3-3 β was incubated with the solubilized P-2 from rat adipocytes treated with or without 3 nmol/l insulin. This resin-bound complex was then subjected to immunoblot analysis with anti-PDE3B antibody raised against a synthetic rat PDE3B peptide (6). GST·14-3-3 β but not GST alone was associated with endogenous PDE3B (Fig. 2B, top). This association was increased in response to insulin. Accordingly, the PDE3B activity in the GST·14-3-3 β resin-bound complex was increased in response to insulin (Fig. 2B, bottom). The amount of PDE3B protein in the solubilized P-2 before GST pull-down was the same in the presence or absence of insulin (as shown in Fig. 2A, top), suggesting that the insulin-stimulated form of endogenous PDE3B associates more effectively with 14-3-3 β than the enzyme from untreated cells.

    We next examined whether endogenous PDE3B associates with endogenous 14-3-3 β in vivo, in rat adipocytes. The solubilized P-2 of rat adipocytes, treated with or without insulin, was immunoprecipitated with the anti-14-3-3 β antibody. The protein G-agarose-bound immune complex was then subjected to immunoblot analysis with anti-PDE3B antibody. The PDE3B protein was only weakly associated with 14-3-3 β in the basal state, but insulin markedly increased this association (Fig. 2C, top). PDE3B activity in the agarose-bound immune complex was increased approximately fourfold by insulin (Fig. 2C, bottom). The reverse experiment was performed to confirm further this association. The solubilized P-2 was first immunoprecipitated with the anti-PDE3B antibody, and the agarose-bound immune complex was then immunoblotted with the anti-14-3-3 β antibody. An insulin-induced increase of the association of the two proteins was consistently detected (data not shown). These in vivo results indicate that PDE3B associates with 14-3-3 β and that this interaction is enhanced by insulin in rat adipocytes.

    The association between PDE3B and 14-3-3 β is serine phosphorylation-dependent.

    Insulin promotes serine phosphorylation and the activation of PDE3B in adipocytes (16). Members of the 14-3-3 protein family are known to bind to a specific phosphoserine-containing motif in various proteins (10, 17). We thus examined whether the insulin-induced association of endogenous PDE3B with endogenous 14-3-3 β is phosphorylation-dependent. Treatment of adipocytes with 1 μmol/l okadaic acid, a potent, cell-permeable Ser/Thr phosphatase inhibitor, increased the association of PDE3B with 14-3-3 β to the same level as that achieved with insulin (Fig. 3A, top). Pretreatment with 1 μmol/l okadaic acid did not further enhance the effect of insulin on this association. Likewise, PDE3B activity in the solubilized P-2 was increased to the same extent by okadaic acid and insulin (Fig. 3A, bottom). Okadaic acid and insulin are reported to exert similar effects on PDE3B phosphorylation (18). In addition, when the solubilized P-2 was prepared in buffer lacking phosphatase inhibitors, both the protein-protein association and insulin-induced PDE3B activation were abolished (data not shown). That the PDE3B synthesized by the reticulocyte lysate system interacts with 14-3-3 β in the absence of insulin (Fig. 1) suggests that this protein is phosphorylated. Indeed, certain proteins synthesized in the system are phosphorylated (19). In general, there seems to be a positive correlation between insulin-induced phosphorylation of PDE3B, activation of the enzyme, and the association of 14-3-3 β with the enzyme. Isoproterenol, another inducer of PDE3B phosphorylation and activation, also promotes the interaction between PDE3B and 14-3-3β (data not shown).

    PI3-K is required for the 14-3-3·PDE3B interaction.

    We have previously reported that 100 nmol/l wortmannin, a relatively specific inhibitor of PI3-K, inhibits insulin-dependent phosphorylation and activation of PDE3B in rat adipocytes (6). We next tested whether wortmannin would prevent the insulin-induced association of endogenous PDE3B with endogenous 14-3-3 β in rat adipocytes. Pretreatment of adipocytes with 100 nmol/l wortmannin completely inhibited the insulin-induced activation of PDE3B and its association with 14-3-3 β (Fig. 3B). In addition, pretreatment of adipocytes with 100 μmol/l Ly294002, another PI3-K inhibitor, had the same effect (data not shown). These results suggest that PI3-K-dependent phosphorylation of PDE3B by insulin is required for the association of 14-3-3 β with PDE3B.

    Specific phosphopeptides block the 14-3-3·PDE3B interaction.

    Insulin is reported to promote the phosphorylation of mouse Ser-273 (rat PDE3B on Ser-279) (8) or rat PDE3B on Ser-302 (7), and cAMP-dependent protein kinase (PKA) results in phosphorylation of rat PDE3B on Ser-427 (20). We therefore examined whether phosphorylation of any of these residues is required for the interaction between endogenous PDE3B and endogenous 14-3-3 β. Τhe effects of synthetic 15 amino acid peptides of rat PDE3B, containing either phosphorylated serine (pS279, pS302, or pS427) or nonphosphorylated serine (S279, S302, or S427) were tested on the protein-protein interaction. The pS279 and pS302 peptides both inhibited the insulin-induced association of PDE3B with 14-3-3 β in rat adipocytes (Fig. 3C). These effects were dose-dependent (data not shown). The corresponding nonphosphorylated peptides (S279 and S302) had no effect. Neither pS427 nor S427 had an effect. Thus, the two insulin-regulated phosphorylation sites, Ser-279 and Ser-302, are involved in the insulin-induced association of 14-3-3 β with PDE3B. β-Adrenergic agonists, as well as insulin, phosphorylate and activate PDE3B (21,22), but the exact residues involved remain controversial. Rahn et al. (7), using 32P-labeled rat adipocytes, reported that insulin and isoproterenol both phosphorylate Ser-302 of rat PDE3B. Kitamura et al. (8), using mutagenesis analysis in 3T3L1 adipocytes, reported that insulin phosphorylates Ser-273 of mouse PDE3B (rat Ser-279), whereas isoproterenol phosphorylates the mouse enzyme on Ser-296 (rat Ser-302). The present data suggest that both Ser-279 and Ser-302 in rat PDE3B are involved in the insulin-induced interaction between 14-3-3 β and PDE3B. However, the addition of phosphorylated peptides (pS279 and pS302) to the solubilized P-2, which results in the dissociation of 14-3-3 β from PDE3B, caused only a slight decrease of insulin-induced PDE3B activity (data not shown). Thus, 14-3-3 β may not directly affect PDE3B activity, although the 14-3-3 protein is reported to regulate directly the phosphorylation and activity of Raf-1 and tyrosine hydroxylase (2325).

    14-3-3 proteins interact with conserved phosphorylated motifs in other proteins. In these motifs, an Arg residue at the -3 or -4 position relative to the phosphoserine is crucial, and Pro at +2 is also important (26). The sequences around Ser-279 and Ser-302 in rat PDE3B both contain Arg at -3 and -4 and the sequence around Ser-302 also includes Pro at +2. PDE3B therefore contains motifs with the potential of interacting with 14-3-3 proteins. In addition, the sequence around Ser-279 on rat PDE3B is consistent with the motif RX1–2SX2–3S found in Cbl and PKCμ, both of which bind to 14-3-3 proteins (27,28).

    In summary, we have demonstrated an interaction between 14-3-3 and PDE3B, a key enzyme in the metabolic action of insulin. This interaction occurs in vivo and in vitro, is insulin-inducible, and involves the phosphorylation on serine residues 279 and 302 in rat PDE3B. 14-3-3 proteins exist as dimers and can simultaneously bind two ligands. The reports that IRS-1 associates with 14-3-3 β raise the possibility that the latter serves as a scaffolding protein to link IRS-1 to PDE3B. It is not clear whether the interaction between these molecules is involved in basal or insulin-mediated antilipolysis. Additional experiments are required to clarify these points.

    FIG. 1.

    PDE3B associates with 14-3-3 β in vitro. The GST pull-down assay was used to detect the association of in vitro synthesized PDE3B with GST·14-3-3 β. The FLAG-tagged full-length or truncated forms of mouse PDE3B, represented as mPDE3B (full-length), mPDE3B (aa 1–322), mPDE3B (aa 223–636), or mPDE3B (aa 621–1,100), were labeled with [35S]methionine. The radiolabeled PDE3B proteins and either GST·14-3-3 β (lanes 6, 8, 10, and 12) or GST (lanes 5, 7, 9, and 11) glutathione resin complex were incubated as described in research design and methods. A sample representing 10% of the input labeled proteins (lanes 1–4) and the entire pull down complexes (lanes 5–12) were subjected to SDS-PAGE. The gel was dried, and 35S-labeled proteins were visualized using a image analyzer. Lanes 1, 5, and 6 represent full-length mouse PDE3B; lanes 2, 7, and 8 represent mouse PDE3B (aa 1–322); lanes 3, 9, and 10 represent mouse PDE3B (aa 223–636); lanes 4, 11, and 12 represent mouse PDE3B (aa 621–1,100). Data are representative of three independent experiments. The smaller product in lane 4 could be a degraded protein, which should not affect the results.

    FIG. 2.

    Insulin induces the association of PDE3B with 14-3-3 β in rat adipocytes. A: The PDE activity in the solubilized P-2 reflects PDE3B activity in rat adipocytes. PDE activity in solubilized P-2 in the presence or absence of cilostamide (1 μmol/l) was measured (bottom). The open bars represent total activity, and the hatched areas represent cilostamide-insensitive PDE activity. The PDE3B in the solubilized P-2 (10 μg of protein) was also quantified by Western blotting using an anti-PDE3B antibody (top). The visualized data are representative of three independent experiments, and quantitative data represent means ± SE from three independent experiments. *Significant difference compared with insulin (−) (P < 0.01). B: Rat adipocyte PDE3B associates with GST·14-3-3 β in vitro. Solubilized P-2 (200 μg of protein) was incubated with GST glutathione resin or GST·14-3-3 β glutathione resin as described in research design and methods. The resin-bound complexes were subjected to SDS-PAGE, and immunoblotting was performed using an anti-PDE3B antibody (top). The amount of the added GST fusion proteins was assessed by amido black staining of the electrotransferred membrane (middle). PDE activity in the pull-down complex was assayed and is shown in the lower data set. The visualized data are representative of three independent experiments, and quantitative data represent means ± SE from three independent experiments. *Significant difference compared with insulin (−), pull-down by GST·14-3-3 β (P < 0.01). C: Rat adipocyte PDE3B associates with endogenous 14-3-3 β in vivo. Solubilized P-2 (200 μg of protein) from rat adipocytes incubated with or without 3 nmol/l insulin was incubated with an anti-14-3-3 β antibody as described in research design and methods. The agarose-bound immune complex was subjected to SDS-PAGE. The amount of PDE3B in the solubilized P-2 (10 μg of protein) before immunoprecipitation was quantified by Western blotting using an anti-PDE3B antibody (top lane). An anti-14-3-3 β antibody was used to assess the amount of the immunoprecipitated 14-3-3 β (middle lane). Immunoblotting was done using the same anti-PDE3B antibody to detect the PDE3B associated with 14-3-3 β (bottom lane). PDE activity in the agarose-bound immune complex coimmunoprecipitated with 14-3-3 β was also measured (bottom). The visualized data are representative of three independent experiments, and quantitative data represent means ± SE from three independent experiments. *Significant difference compared with insulin (−) (P < 0.01).

    FIG. 3.

    The association of PDE3B with 14-3-3 β is phosphorylation dependent. A: Effect of okadaic acid on the association of endogenous PDE3B with endogenous 14-3-3 β in rat adipocytes. Isolated rat adipocytes were incubated in the presence or absence of okadaic acid (1 μmol/l) at 37°C for 20 min, and then with or without insulin (3 nmol/l) for 10 min. The PDE3B in the solubilized P-2 (10 μg of protein), before immunoprecipitation, was quantified by Western blotting (top lane). The solubilized P-2 (200 μg of protein) was also subjected to immunoprecipitation using the anti-14-3-3 β antibody. The immune complexes were subjected to immunoblot analysis using the anti- 14-3-3 β antibody (middle lane) or anti-PDE3B antibody (bottom lane). PDE activity was also measured in the solubilized P-2 before immunoprecipitation (bottom). The visualized data are representative of three independent experiments, and quantitative data represent the means ± SE of three independent experiments. *Significant difference compared with insulin (−)/okadaic acid (−) control (P < 0.01). B: Effect of wortmannin on the association of endogenous PDE3B with endogenous 14-3-3 β in rat adipocytes. Isolated rat adipocytes were incubated in the presence or absence of wortmannin (100 nmol/l) at 37°C for 30 min and then with or without insulin (3 nmol/l) for 10 min. The PDE3B in the solubilized P-2 (10 μg of protein) before immunoprecipitation was quantitated by Western blotting (top lane). Solubilized P-2 (200 μg protein) was also immunoprecipitated with anti-14-3-3 β antibody. The immune complexes were subjected to immunoblotting with anti-14-3-3 β antibody (middle lane) or anti-PDE3B antibody (bottom lane). PDE activity was measured in the solubilized P-2 before immunoprecipitation (bottom). The visualized data are representative of three independent experiments and quantitative data represent the means ± SE of three independent experiments. Significant difference compared with wortmannin (−), insulin (−) (P < 0.01). †Significant difference compared with wortmannin (−), insulin (+) (P < 0.01). C: Serine phosphorylated peptides inhibit the association of endogenous PDE3B with endogenous 14-3-3 β in rat adipocytes. Ser-phosphorylated (pS279, pS302, and pS427) or nonphosphorylated (S279, S302, and S427) peptides, each of which consists of 15 amino acids corresponding to the sequence around Ser-279, Ser-302, and Ser-427 in rat PDE3B, respectively, were synthesized. Rat adipocytes were incubated with or without 3 nmol/l insulin at 37°C for 10 min and solubilized P-2 was obtained. They (200 μg of protein) were then incubated at 4°C overnight with or without one of the peptides (1 mmol/l) and immunoprecipitated with anti-14-3-3 β antibody. The PDE3B in solubilized P-2 (10 μg of protein) was quantified by Western blotting (top lane). The immune complexes were subjected to immunoblot analysis using the anti-14-3-3 β antibody (middle lane) or anti-PDE3B antibody (bottom lane). Note: The Ser-279, Ser-302, and Ser-427 in rat PDE3B correspond to Ser-273, Ser-296, and Ser-421 in mouse PDE3B. The data are representative of three independent experiments.

    Acknowledgments

    This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) “Medical Genome Science” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 12204007), a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (Nos. 11671122 and 11671124), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (No. 60206-00).

    We thank Drs. Philip James, Alan Saltiel, and Masato Kasuga for providing PJ69-4A, 3T3L1 adipocyte cDNA libraries, and the mouse PDE3B cDNA, respectively. We also thank Drs. Akira Sakai and Minako Kakui for technical advice in the use of two-hybrid screening.

    Footnotes

    • Address correspondence and reprint requests to Dr. H. Osawa and Dr. H. Makino, Department of Laboratory Medicine, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan. E-mail: harosawa{at}m.ehime-u.ac.jp, hidemak{at}m.ehime-u.ac.jp.

      Received for publication 7 April 2002 and accepted in revised form 23 August 2002.

      β-gal, β-galactosidase; GST, glutathione S-transferase; IRS, insulin receptor substrate, PDE, phosphodiesterase; PI3-K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride.

    REFERENCES

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