Cyclic Nucleotide 3B Is a Downstream Target of Protein B and May Be Involved in Regulation of Effects of on Thymidine This information is current as Incorporation in FDCP2 Cells of September 25, 2021. Faiyaz Ahmad, Li-Na Cong, Lena Stenson Holst, Ling-Mei Wang, Tova Rahn Landstrom, Jaclyn H. Pierce, Michael J. Quon, Eva Degerman and Vincent C. Manganiello

J Immunol 2000; 164:4678-4688; ; Downloaded from doi: 10.4049/jimmunol.164.9.4678 http://www.jimmunol.org/content/164/9/4678 http://www.jimmunol.org/ References This article cites 84 articles, 48 of which you can access for free at: http://www.jimmunol.org/content/164/9/4678.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells1

Faiyaz Ahmad,2* Li-Na Cong,† Lena Stenson Holst,‡ Ling-Mei Wang,§ Tova Rahn Landstrom,‡ Jaclyn H. Pierce,§ Michael J. Quon,† Eva Degerman,‡ and Vincent C. Manganiello*

Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba؊, F/Bb؊, F/Bc؊) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of -like growth factor-1 (IGF-1), activities of PKB,

cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) Downloaded from cells, and F/B and F/B؊ cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities ϳ2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity ϳ10-fold and PDE3B and activity (ϳ4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased (ϳ10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B؊ cells, IGF-1 activated PDE4, not PDE3B, suggesting that

kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in http://www.jimmunol.org/ F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells. The Journal of Immunology, 2000, 164: 4678–4688.

he /threonine protein kinase PKB (protein kinase B)3 There is considerable interest in identifying downstream targets (also known as Akt or RAC-PK) is a downstream target and effectors of PKB action. Glycogen synthase kinase was the T and effector of phosphatidylinositol-3 kinase (PI3-K) ac- first physiological substrate identified (12). Cardiac 6-phospho- by guest on September 25, 2021 tion, and is thought to mediate many metabolic, mitogenic, and fructo-2 kinase, a critical in the regulation of glycolysis, antiapoptotic effects of insulin, IGF-1, IL-3, and other growth fac- was also reported to be phosphorylated and activated by PKB tors and cytokines (1–3). For example, PKB has been implicated in vitro (13). BAD, a member of the Bcl-2 family that promotes in insulin-stimulated translocation of GLUT4 transporter and cell death, is phosphorylated in intact cells and in vitro by PKB. transport in rat adipocytes (4, 5), insulin stimulation of IL-3-induced phosphorylation of BAD is thought to result in its glycogen synthesis (6, 7), meiotic maturation of frog oocytes functional inactivation by promoting its binding to 14-3-3 (10). (8), and antiapoptotic actions of IGF-1 (9) and IL-3 (10, 11). PKB is also thought to promote cell survival by phosphorylating Forkhead family transcription factors, resulting in their association with 14-3-3 proteins, retention in the , and functional inac- tivation (14). In endothelial cells, PKB increases NO production *Pulmonary/Critical Care Medicine Branch and †Hypertension-Endocrine Branch, and release by phosphorylation and activation of endothelial NO National , Lung, and Blood Institute, and §Laboratory of Cellular and Molecular synthase (15). Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and ‡Section for Molecular Signaling, Department of Cell and Molecular More recently, in oocytes (8), rat adipocytes (16, 17), and Biology, Lund University, Lund, Sweden 3T3-L1 adipocytes (18), PDE3 isoforms have been suggested to Received for publication June 17, 1999. Accepted for publication February 11, 2000. be downstream targets and effectors of PKB actions. In adipo- The costs of publication of this article were defrayed in part by the payment of page cytes, insulin-induced activation of PDE3B, via P13-K- and charges. This article must therefore be hereby marked advertisement in accordance PKB-dependent signals, is a critical component in the antilipo- with 18 U.S.C. Section 1734 solely to indicate this fact. lytic action of insulin (19–21). Activation of PDE3 by PKB is 1 E.D. was supported by Medical Research Council Grant (Sweden) 3362. thought to be involved in the resumption of meiosis in quiescent 2 Address correspondence and reprint requests to Dr. Faiyaz Ahmad, PCCMB, Na- tional Heart, Lung, and Blood Institute, Building 10, Room 5N-307, 10 Center Drive, frog oocytes (8). MSC 1434, Bethesda, MD 20892-1434. E-mail address: [email protected] FDCP2 promyeloid cells have been utilized to study the mito- 3 Abbreviations used in this paper: PKB, protein kinase B; 8-Br-cAMP, 8-bromo- genic actions of insulin and IL-4, and to establish the role of in- cAMP; CT, carboxyl terminus; GSK-3, glycogen synthase kinase-3; IGF-1, insulin- sulin receptor-substrate proteins (IRS-1, IRS-2) in these processes like growth factor-1; IRS, insulin receptor substrate; MAP, mitogen-activated protein; MBP, myelin basic protein; ERK, extracellular signal-related kinase; MEK, MAP/ (22). In these cells, activation of insulin, IGF-1, or IL-4 receptors ERK kinase; MPDE3B, mouse PDE3B; NT, amino terminus; PDE, cyclic nucleotide generates some common signals (23, 24). FDCP2 cells require phosphodiesterase; PDK, phosphoinositide-dependent kinase; PI3-K, phosphatidyl- inositol 3-kinase; PKA, cAMP-dependent ; RPDE3B, rat PDE3B; wt, IL-3 for growth; IL-4 cannot replace IL-3, but enhances its effects wild-type. (25). In FDCP2 cells, IL-3 and IL-4 interact with different types of

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 4679 receptors and induce tyrosine phosphorylation of different ␮M selenium, and 1 mg/ml BSA), and incubated (3 ml cells/well, ϳ7 ϫ proteins. For example, IL-4, not IL-3, stimulates tyrosine phos- 105 cells/ml) in six-well Costar plates (Cambridge, MA) (25). After 2–3 h phorylation of IRS-2 (25, 26). FDCP2 cells also contain PDE4 as at 37°C, IGF-1 or cytokines such as IL-3 were added as indicated. Finally, cells were harvested by centrifugation (1200 ϫ g, 5 min), and suspended well as PDE3B, and recently we found that in these cells, IL-4 and homogenized (10–15 strokes in a Dounce homogenizer (Kontes In- activates both PDE3B and PDE4, whereas IL-3 and PMA selec- struments, Vineland, NJ)) in lysis buffer A (20 mM Tris, pH 7.5, 150 mM tively activate PDE4 (26). IL-4, IL-3, and PMA activate PDE4 via NaCl, 1 mM MgCl2, 1% Nonidet P-40 detergent, 1 mM EDTA, 10 mM mitogen-activated protein kinase (MAP kinase)-dependent signals, NaF, 10 mM sodium pyrophosphate, 1 mM Na3V04, 1 mM PMSF (Sigma, St. Louis, MO), 10 ␮g/ml each of aprotinin, pepstatin, and leupeptin whereas, similar to insulin in rat adipocytes (16, 17, 21), IL-4 (Boehringer Mannheim, Indianapolis, IN) and 1 mM benzamidine (Sigma), activates PDE3B via MAP kinase-independent signals (26). and kept for 30 min at 4°C. Protein was measured using the Bradford assay The goal of this study was to evaluate the role of PKB in the (Bio-Rad, Richmond, CA) with BSA as standard. activation of PDE3B by IGF-1 in FDCP2 cells and to determine Expression of PKB in FDCP2 cells whether PDE3B was a downstream effector and regulator of some PKB actions. For this purpose, we generated permanently trans- The following plasmid vector constructs (4, 27) were transformed into fected FDCP2 cells that overexpress wt (F/B), constitutively active Escherichia coli, amplified, and purified. (F/B*), and three kinase-inactive (F/BaϪ, F/BbϪ, F/BcϪ) forms of pCIS2 (F/V). A vector that generates high levels of expression (27) was used as parent vector for subsequent constructions. PKB (5, 27). In the absence of IGF-1, activities of PKB, PDE3B, Akt-WT (F/B). A 1.4-kb XbaI/BamHI fragment containing the cDNA and PDE4 were similar in nontransfected FDCP2 cells, cells trans- for mouse Akt-1 (28) was blunt ended and ligated in the sense orientation fected with control vector (F/V), and F/B cells. In F/V cells, IGF-1 into the HpaI site in the multiple cloning region of pCIS2. increased PKB, PDE3B, and PDE4 activities ϳ2-fold. In F/B cells, Constitutively active Akt-myrA (F/B*). A BglII/BamHI fragment Downloaded from however, IGF-1, in a wortmannin-sensitive manner, increased containing the cDNA for mouse Akt-1 with a myristoylation sequence PKB ϳ10-fold and PDE3B activity (ϳ4-fold) and phosphoryla- from pp60 c-src (29) fused in-frame with the N terminus of Akt was blunt tion, but increased PDE4 only ϳ2-fold (as in F/V cells). In F/B* ended and ligated in the sense orientation into the HpaI site in the multiple cloning region of pCIS2. cells, in the absence of IGF-1, PKB was increased ϳ10-fold and ؊ Kinase-inactive Akt (F/B ). A point mutant of Akt-WT with a sub- PDE3B was activated (3–4-fold) and phosphorylated. Wortman- stitution of alanine for lysine at position 179 in the canonical ATP binding Ϫ nin inhibited activation of PKB and phosphorylation/activation of site (F/Ba ) was constructed using the mutagenic oligonucleotide-1 http://www.jimmunol.org/ PDE3B. In F/B* cells, IGF-1 had little further effect on PKB and (5Ј-GC TAC TAT GCC ATG GCG-ATG CTC AAG AAG-G-3Ј) and the activation/phosphorylation of PDE3B, and increased PDE4 to the MORPH site-specific plasmid DNA mutagenesis kit, according to the man- ufacturer’s instructions (5 Prime 3 3 Prime, Boulder, CO). The mutation same extent as in F/V cells. In cells transfected with kinase-inac- introduced a NcoI site and was confirmed by direct sequencing. Substitu- tive forms of PKB, IGF-1 activated PDE4, not PDE3B, suggesting tions of alanine for threonine at position 308 and alanine for serine that kinase-inactive PKB functioned as a dominant negative with at position 473 (phosphorylation sites in the regulatory region of Akt) Ϫ respect to activation of PDE3B. Activated PKB phosphorylated (F/Bb ) were constructed using oligonucleotide-2 (5Ј-CCA CTA TGA Ј Ј and activated rPDE3B in vitro. Proliferation of F/B* cells and AGG CAT TTT GCG GAA CGC CGG-3 ) and oligonucleotide-3 (5 -TTC CCC CAG TTC GCC TAC TCG GCC AGT GGC ACA-3Ј). In addition to IGF-1-induced phosphorylation of BAD in F/B cells was more introducing the changes noted above, the mutagenic oligonu- sensitive to inhibition by PDE3 inhibitors than the PDE4 inhibitor cleotides also introduced silent mutations that destroyed an XmnI site by guest on September 25, 2021 rolipram. These and other results are consistent with the idea that and created a new BglI site. The presence of the correct mutations was in FDCP2 cells, PDE3B, not PDE4, is a target, if not substrate, of confirmed by direct sequencing. Another kinase-inactive mutant AktAAA (F/BcϪ) was created by substitution of alanine for lysine at position 179, activated PKB and a downstream effector of some actions medi- alanine for threonine at position 308, and alanine for serine at position 473, ated by PKB. as described above. Expression vectors for the various constructs described above and a Materials and Methods plasmid containing the neomycin resistance gene (ϳ20 ␮g total DNA) were introduced into FDCP2 cells by electroporation; transformants (F/V, Transferrin and selenium were purchased from Collaborative Research F/B, F/B*, and F/Ba,b,cϪ) were selected by growing cells in 24-well cul- (Boston, MA); [3H]cAMP, from New England Nuclear (Boston, MA); anti- ture plates in medium with G418 (750 mg/L), as previously described (22, human PKB-CT, anti-mouse BAD-NT, and anti-rat p70S6 kinase poly- 24, 30). Six different colonies of each construct were isolated; experiments clonal Abs, from Santa Cruz Biotechnology (Santa Cruz, CA); anti-human were conducted with at least two independent colonies. FDCP2 cells ex- extracellular signal-related kinase (ERK1) (MAP kinase) mAb, from Sig- pressing similar amounts of wt (F/B), constitutively active (F/B*), and nal Transduction Laboratories (Lexington, KY); anti-rat ERK1-CT poly- kinase-inactive PKB (F/BaϪ, F/BbϪ, F/BcϪ) were selected by analyzing clonal (MAP kinase) Ab, from Upstate Biotechnology (Lake Placid, NY); lysates (30–40 ␮g protein) from equivalent numbers of cells by SDS- the PI3-K inhibitor wortmannin, from Biomol Research Laboratories (Ply- PAGE (Novex, San Diego, CA), and immunoblotting with PKB-CT Ab mouth Meeting, PA); MEK-1 inhibitor PD98059, from New England Bio- (Fig. 1). PKB activity was also determined in these lysates. labs (Beverly, MA); phosphorus-32 as H 32PO (1000 Ci/mmol) and 3 4 3 [␥-32P]ATP (3000 Ci/mmol), from ICN Radiochemicals (Costa Mesa, Incorporation of [ H]thymidine into DNA in FDCP2 cells CA); and [3H]thymidine (22 Ci/mmol), from Amersham (Arlington Heights, IL). K9 (KKRNRTLTK) and Crosstide (GRPRTSSFAEG) pep- Thymidine incorporation was assayed as previously described (22). Cells in log phase growth were washed twice in PBS and suspended in growth tides were synthesized at the Biomolecular Unit, Lund University (Lund, ϫ 4 Sweden). Prestained molecular size markers were from Bio-Rad (Rich- medium; 3 10 cells were added to each of 24 wells (final volume, 1 ml) mond, CA). Other materials were obtained as indicated and were of the containing RPMI 1640 medium with or without conditioned medium con- highest grade available. taining IL-3 (5% WEHI) and the indicated concentrations of PDE inhibi- tors, 8-Br-cAMP, or PGE1. DMSO or ethanol at a final concentration of Cell culture and incubations 0.1% or less was added to appropriate wells as vehicle controls. Cells were grown for 48 h at 37°C. [3H]Thymidine (22 Ci/mmol; Amersham) was The murine, IL-3-dependent hemopoietic cell line, FDCP2, was propagated added to a final concentration of 0.5 ␮Ci/ml, and incubation was continued and maintained in RPMI 1640 medium (Life Technologies, Grand Island, for 2 h. Cells were harvested (Skatron Cell Harvester) and lysed on glass NY) supplemented with 10% FBS, 5% WEHI-3B (American Type Culture microfiber filters; unincorporated nucleotide was removed by repeated Collection, Rockville, MD)-conditioned medium (which contains IL-3), washing with water. Filters were dried and counted in scintillation fluid. and 2 mM glutamine (25). For most experiments, exponentially growing Data are presented as mean Ϯ SEM of cpm in samples from triplicate FDCP2 cells (2–5 ϫ 105 cells/ml) were collected, centrifuged (5 min, incubations. For cell survival assays, IL-3-depleted cells were seeded at a 1200 ϫ g), washed twice, and cultured overnight in RPMI 1640/10% FBS. density of 3 ϫ 104/ml in RPMI 1640 containing 10% FBS. In some ex- Immediately before experiments, cells were washed twice, suspended in periments, the ratio of viable to dead cells was determined by trypan blue serum-free RPMI 1640 medium containing TSB (5 ␮g/ml transferrin, 10 exclusion. 4680 PDE3B IS A TARGET AND EFFECTOR OF PKB ACTION

Table I. Effects of IGF-1 on PDE3 and PDE4 activities in FDCP2 cellsa

PDE Activity (pmol cAMP hydrolyzed/min/mg)

ϪIGF-1 ϩIGF-1

Cells Total PDE PDE3 PDE4 Total PDE PDE3 PDE4

F(n ϭ 8) 12.5 Ϯ 1.0 5.7 Ϯ 0.7 6.2 Ϯ 1.1 21.0 Ϯ 2.3 11.0 Ϯ 1.7 10.8 Ϯ 0.5 F/V (n ϭ 9) 13.4 Ϯ 1.3 5.9 Ϯ 0.9 6.6 Ϯ 1.1 24.6 Ϯ 4.7b 12.9 Ϯ 2.9b 11.8 Ϯ 2.5b F/B (n ϭ 8) 14 Ϯ 2.6d 6.1 Ϯ 1.4d 7.1 Ϯ 1.5d 4.1 Ϯ 8b 25.5 Ϯ 4.4b 12.7 Ϯ 2.4b F/BaϪ (n ϭ 3) 13.8 Ϯ 0.8d 5.9 Ϯ 0.6d 6.1 Ϯ 0.9d 18.9 Ϯ 0.4b 6.2 Ϯ 1.3d 10.9 Ϯ 0.6c F/BbϪ (n ϭ 3) 13.4 Ϯ 1.3d 5.9 Ϯ 0.5d 6.5 Ϯ 0.5d 22.1 Ϯ 1.5b 6.0 Ϯ 1.0d 13.2 Ϯ 0.7b F/BcϪ (n ϭ 3) 13.4 Ϯ 1.0d 6.0 Ϯ 0.9d 6.6 Ϯ 0.4d 21.0 Ϯ 1.5b 5.8 Ϯ 0.3d 12.3 Ϯ 0.9b F/B* (n ϭ 10) 32.5 Ϯ 5.2b 21 Ϯ 3.7b 8.3 Ϯ 2.1c 32.5 Ϯ 1.9b 21.1 Ϯ 1.9b 10.9 Ϯ 1.0c

a Cells were incubated without or with 10 nM IGF-1 for 10 min before total PDE and PDE3 and PDE4 activities in cell lysates were assayed as described in Materials and Methods. PDE3 activity represents cAMP hydrolysis inhibited by cilostamide (a selective PDE3 inhibitor); PDE4, that activity inhibited by rolipram (a selective PDE4 inhibitor). PDE activities are reported as pmol cAMP hydrolyzed/min/mg (mean Ϯ SEM) from the indicated number (n) of experiments and include data from Figs. 2–4. b p Ͻ 0.001; c p Ͻ 0.02; d p Ͼ 0.05, compared to activities in F/V cells. PDE activities in nontransfected FDCP2(F) cells were not significantly different from those in F/V cells. Downloaded from cAMP PDE assay ml) in six-well Costar plates (25) for 1–2 h at 37°C. Cells were then in- ␮ ␮ cubated in RPMI 1640 medium containing 300 MKH2PO4 and 1% BSA Samples of cell lysates (usually 100 l) were incubated for 10 min at 30°C with or without 32PO (40 ␮Ci/ml) for 90–100 min at 37°C, and finally in a total volume of 0.3 ml containing 50 mM HEPES, pH 7.5, 8.3 mM 4 with or without 10 nM IGF-1 for 10 min. PDE3B and BAD were immu- MgCl , 0.1 mM EDTA, and 0.1 ␮M[3H]cAMP (25–35,000 cpm) as sub- 2 noprecipitated from cleared lysates with anti-PDE3B-NT or anti-BAD-NT strate (31). After dephosphorylation of [3H]5-AMP to [3H]adenosine with Crotalus atrox venom (Sigma), the product was separated from substrate Abs, respectively. SDS-PAGE was then conducted, and dried gels were scanned on a PhosphorImager (Molecular Dynamics), as described above. http://www.jimmunol.org/ by ion-exchange chromatography (QAE-Sephadex; Pharmacia, Piscat- 32 away, NJ) and quantified by scintillation counting. Lysates were diluted so Immunoprecipitates from cells incubated without PO4 were prepared that hydrolysis of substrate was usually less than 20%. PDE3 activity is that similarly and immunoblotted with anti-PDE3B or anti-BAD Abs. PKB fraction of total activity inhibited by 0.3 ␮M cilostamide, a specific PDE3 activity was assessed by phosphorylation of Crosstide in PKB inhibitor, and PDE4 activity, that inhibited by 0.5 ␮M rolipram, a specific immunoprecipitates. PDE4 inhibitor (32). Inhibitor vehicle (DMSO), added in equal quantities to samples without inhibitor, did not alter PDE activities. PKB assays with Crosstide, K9 peptide, and histone 2B substrates Immunoprecipitation and immunoblotting Immunoprecipitation with anti-PKB-CT Ab (50 ␮l/10 ␮g IgG) was con- For most experiments, portions of FDCP2 lysates (2–3 mg protein) were

ducted for2hatroom temperature or overnight at 4°C, as described above, by guest on September 25, 2021 ␮ first cleared by incubation with 1 g of preimmune mouse IgG for1hat and washed proteins were suspended in 30–40 ␮l of reaction buffer (20 room temperature, and then with 60 ␮l of protein G-Sepharose (Pharmacia ␮ ϫ mM HEPES, pH 7.4, 1 mM DTT, 10 mM MnCl2, 10 mM MgCl2,5 M Biotech, Uppsala, Sweden) for 30 min before centrifugation (2800 g, ATP, 10 ␮Ci [␥-32P]ATP, 5 ␮M cAMP protein kinase inhibitor) containing 4°C, 5 min). Cleared cell lysates were incubated with specified Abs for 2 h either 13 ␮g of K9 peptide (KKRNRTLTK), 1 ␮g of Crosstide at room temperature, followed by incubation with fresh protein G-Sepha- (GRPRTSSFAEG), or 2.5 ␮g of histone-2B (Boehringer Mannheim) as rose for 30 min before centrifugation (2800 ϫ g, 4°C, 5 min). Immuno- precipitates were washed three times with lysis buffer A containing 0.1% substrate (17, 35, 36). As recently described (26), after incubation for 15 Nonidet P-40, boiled in Laemmli buffer (Bio-Rad, Richmond, CA) (33), min at 30°C, assay reactions with K9 or Crosstide peptides were terminated ␮ ␮ and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose by addition of 10 l of 1% BSA, 1 mM ATP, pH 3, and 5 l of 30% TCA. membranes in Tris-glycine buffer (25 mM Tris-base and 192 mM glycine For reaction blanks, the stopping solution was added to immunoprecipi- at pH 8.3), containing 20% methanol. Membranes were incubated in blot- tated samples before assay buffer. Samples were centrifuged, and 25 ␮lof supernatant was applied to phosphocellulose (P81; Whatman, Tewksbury, ting buffer (150 mM NaCl, 0.05% (v/v) Nonidet P-40, 0.01% NaN3, and 10 mM Tris, pH 7.4), containing BSA (50 mg/ml) and OVA (10 mg/ml), for MA) paper (2 ϫ 2-cm squares), which were washed three times (5 min 1 h at room temperature with rocking, and then for an additional2hinthe each) with phosphoric acid (7.5 mg/ml), and once with acetone (5 min) same solution containing the appropriate Abs. Membranes were washed before radioassay of 32P incorporated into substrate. three times (10 min each) in blotting buffer without Abs, followed by With histone 2B as substrate for PKB, reactions were terminated by incubation with 125I-labeled protein A or protein G (2 ␮Ci) (Amersham addition of Laemmli buffer and boiling. Proteins were separated by SDS- Life Sciences, Arlington Heights, IL) for1hatroom temperature, followed PAGE, the gel was dried, and 32P-labeled histone 2B was detected and by three washes (10 min each) with blotting buffer. Immunoreactive pro- quantified by PhosphorImager (Molecular Dynamics), as recently de- teins were visualized by PhosphorImager analysis (Molecular Dynamics, scribed (26). Sunnyvale, CA). For some immunoblots, instead of 125I-labeled protein A, HRP-labeled MAP kinase and p70S6 kinase assays second Ab (mouse, rat, rabbit, or goat) and enhanced chemiluminescence (ECL) reagents 1 and 2 (Amersham) were used; immunoreactive proteins Activation of MAP kinase was assessed as recently described (26) by mea- were visualized by exposing nitrocellulose filters to Bio-max Kodak film suring phosphorylation of myelin basic protein (MBP) peptide in immu- and developed in a Standard Medical Imaging (Columbia, MD) developer. noprecipitates prepared with anti-ERK1-CT Ab (21 ␮l/10 ␮g IgG; Upstate Biotechnology). Samples of immunoprecipitates were incubated at 30°C Phosphorylation of PDE3B and BAD for 15 min in 50 mM Tris (pH 7.5), 0.4 mM EGTA, 0.4 mM Na3VO4,25 ␮ ␮ ␥ 32 ␮ PDE3B or BAD proteins were identified in FDCP2 cell lysates by immu- mM MgCl2, 150 M ATP, 10 Ci [ - P]ATP (10 Ci/mmol), 5 M cAMP ␮ noprecipitation/immunoblotting with anti-PDE3B-NT or anti-PDE3B-CT protein kinase inhibitor (Calbiochem, San Diego, CA), and 5 M MBP Abs (rabbit Abs raised against peptides derived from deduced sequences in peptide (APRTPGGRR) substrate (Upstate Biotechnology), in final vol- ␮ the N-terminal (RKDERERDTPAMRSPPP, aa 2–18) and C-terminal ume of 50 l. Reactions were terminated, and phosphocellulose squares (NKLQVDNASLPQADE, aa 1078–1092) portions of rat (R)PDE3B) (34), were washed and analyzed, as described for PKB assays. or anti-BAD-NT Ab, respectively. Immediately before experiments, p70S6 kinase was detected in lysates from F/V and F/B* cells after FDCP2 cells transfected with wt (F/B) or constitutively active (F/B*) PKB, separation of proteins (ϳ30 ␮g) by SDS-PAGE and immunoblotting with or vector alone (F/V) were washed twice, suspended in serum-free RPMI polyclonal p70S6 kinase Ab. Activated kinase exhibited a lower electro- 1640 medium containing TSB, and incubated (3 ml cells, ϳ7 ϫ 105 cells/ phoretic mobility than inactive kinase. The Journal of Immunology 4681

FIGURE 1. Effects of IGF-1 on PKB and MAP ki- nase activities in transfected FDCP2 cells. FDCP2 cells, transfected with empty vector (F/V) or wt PKB (F/B), constitutively active (F/B*), and kinase-inactive (F/BaϪ, F/BbϪ, F/BcϪ) forms of PKB, were incubated with or without 10 nM IGF-1 for 10 min. A and D, Proteins (ϳ30 ␮g) in lysates of control and transfected cells were separated by SDS-PAGE and immunoblot- ted with anti-PKB-CT Ab. PKB activity was assessed by phosphorylation of (B and E) Crosstide (mean cpm Ϯ SEM, n ϭ 3) or histone 2B (not shown) with PKB immunoprecipitates from lysates (ϳ2.5 mg pro- tein) of control and IGF-1-stimulated cells. C and F, MAP kinase activity was assessed by phosphorylation of MBP peptide (mean cpm Ϯ SEM, n ϭ 3) with MAP kinase immunoprecipitates from cell lysates (ϳ2.5 mg protein). In this and all other figures, when not present, SEMs were too small to be displayed graphically. Downloaded from

Results and Discussion inant negative with regard to activation of PKB (Fig. 1E) and PDE3B (Table I). In the absence of IGF-1, in FDCP2 cells trans-

PDE3 and PDE4 exhibit high affinity for cAMP (31, 32) http://www.jimmunol.org/ fected with constitutively active PKB (F/B* cells), PDE3B activity and, as shown in Table I, with 0.1 ␮M[3H]cAMP as substrate, was increased ϳ4-fold and similar to that in F/B cells exposed to account for most of the cAMP hydrolytic activity in FDCP2 cell IGF-1. PDE3B activity was not increased further by IGF-1, which lysates (26). Two PDE3 isoforms, PDE3A and PDE3B, have been increased PDE4 in F/B* cells to almost the same extent as in F/B identified (16); PDE3B mRNA was amplified by RT-PCR from or F/V cells (Table I). These results indicate that PKB is an up- FDCP2 cell RNA (31) (data not shown). PDE3B protein in FDCP2 stream regulator of PDE3B, not PDE4, in FDPC2 cells. lysates was identified by immunoprecipitation/immunoblotting As shown in Fig. 1, C and F, MAP kinase was not increased in with anti-PDE3B-NT Ab (34) (data not shown). cells transfected with PKB/Akt constructs. IGF-1 activated MAP kinase ϳ2-fold in all cells, including F/B* cells, indicating that,

Characterization of FDCP2 cells transfected with PKB by guest on September 25, 2021 constructs even in cells overexpressing constitutively activated PKB, MAP kinase was regulated appropriately by IGF-1. As shown in Fig. 2, Several independently isolated lines were generated from a single the MEK-1 inhibitor PD98059 did not alter PDE3B (E) or PKB (F) transfection of FDCP2 cells with constructs that expressed vector activities in F/B* cells, consistent with the observations that MAP alone (F/V) and wt (F/B), constitutively active (F/B*), and three Ϫ Ϫ Ϫ kinase was not elevated in F/B* cells (Figs. 1C and 2D). In non- kinase-inactive (F/B a ,b ,c ) forms of Akt/PKB (Fig. 1). Im- transfected FDCP2 cells, IL-4-induced activation of PDE3B was munoreactive PKB was much higher in the cells transfected with independent of MAP kinase or p70S6 kinase activity, whereas PKB constructs than in F/V cells (Fig. 1, A and D). In the absence IL-4-induced activation of PDE4 was MAP kinase dependent and of IGF-1, PKB activity was similar in F/V and F/B cells, but mark- blocked by PD98059 (26). As also shown in Fig. 2A, in F/B* cells ϳ edly higher ( 10-fold) in F/B* cells, and was affected only (as in other cells transfected with constitutively active PKB (8, 37, slightly by incubation of these latter cells with IGF-1 (Fig. 1, B and 38)), p70S6 kinase was activated, evidenced by the shift in its ϳ E). Incubation with IGF-1 resulted in 2–3-fold activation of PKB mobility on SDS-PAGE. Rapamycin blocked activation of p70S6 ϳ in F/V cells, but 10-fold in F/B cells, to levels comparable with kinase without affecting PKB or PDE3B activity, but did reduce those in F/B* cells (Fig. 1B). IGF-1 had little effect on PKB ac- Ϫ Ϫ Ϫ PDE4 activity, which was slightly increased in F/B* cells (Table I; tivity in F/Ba , F/Bb , F/Bc cells, whether assessed as phos- Fig. 2B). Although this slight increase in PDE4 activity was not phorylation of histone 2B (data not shown) or of Crosstide by PKB observed in all experiments with F/B* cells, these results are con- immunoprecipitates (Fig. 1E). sistent with the findings of Mackenzie et al. (39), which indicated Role of PKB in IGF-I-induced activation of PDE3B in FDCP2 that, in 3T3-F442A cells, PDE4A was activated by p70S6 kinase. cells In untransfected FDCP2 cells, however, effects of IL-3 and IL-4 on PDE4 were blocked by PD98059, not rapamycin (26). In the absence of IGF-1, activities of PDE3B and PDE4 were similar in nontransfected FDCP2 cells, F/V cells, F/B cells, and F/BaϪ, F/BbϪ, and F/BcϪ cells (Table I). Incubation for 10 min Role of PKB in phosphorylation/activation of PDE3B in FDCP2 with 10 nM IGF-1 increased PDE3B and PDE4 activities (Յ2- cells fold) in nontransfected FDCP2 cells as well as in F/V cells. As we We next evaluated the effects of IGF-1 and PKB on phosphoryla- have recently reported, IL-4 also increased PDE3B and PDE4 ac- tion/activation of PDE3B in 32P-labeled FDCP2 cells. As shown in tivities Յ 2-fold in nontransfected FDCP2 cells (26). In F/B cells, Fig. 3, in F/B cells overexpressing wt PKB, neither PDE3B (C) nor however, IGF-1 increased PDE3B by ϳ4-fold, whereas the in- PKB (D) was activated in the absence of IGF-1, and PDE3B was crease in PDE4 activity was similar to that in F/V cells (Յ2-fold). not phosphorylated (A). As shown in Fig. 3, however, after incu- In F/BaϪ, F/BbϪ, and F/BcϪ cells, IGF-1 activated PDE4, not bation of F/B cells with 1 or 10 nM IGF-1 for 10 min, phosphor- PDE3B, suggesting that kinase-inactive PKB functioned as a dom- ylation of PDE3B was markedly increased (A), as was PDE3B 4682 PDE3B IS A TARGET AND EFFECTOR OF PKB ACTION

FIGURE 2. Effect of rapamycin and PD98059 on PDE, PKB, MAP kinase, and p70S6K activities in F/V and F/B* cells. FDCP2 cells, transfected with vector alone (F/V) or constitutively active PKB (F/B* cells), were incubated with or without 20 nM rapamy- cin (rap) (A–C)or10␮M PD98059 (PD) (D–F) for 30 min. A, Lysates (ϳ30 ␮g protein) from F/V and F/B* cells were separated on SDS-PAGE and immunoblot- ted with anti-p70S6K Ab. D, MAP kinase activity (mean cpm Ϯ 1⁄2 range, n ϭ 2) in F/V and F/B* cells was assessed by phosphorylation of MBP peptide with MAP kinase immunoprecipitates from cell lysates (ϳ2.5 mg protein). B and E, Portions of lysates from cells incubated with or without rapamycin (B)or Downloaded from PD98059 (E) were assayed for total PDE (Ⅺ) and PDE3 (u) and PDE4 (f) activities (mean cpm Ϯ 1⁄2 range, n ϭ 2). C and F, Samples of cell lysates (ϳ2.5 mg protein) were immunoprecipitated with anti- PKB-CT Ab; PKB activity (mean cpm Ϯ 1⁄2 range, n ϭ 2) was assessed by phosphorylation of Crosstide by PKB immunoprecipitates. http://www.jimmunol.org/ by guest on September 25, 2021 activity (3–4-fold) (C) and PKB activity (ϳ6–10-fold) (D). In F/V Because phosphoinositide-dependent (PDK) are thought cells, IGF-1 increased PDE3B and PDE4 activities ϳ2-fold (Fig. to phosphorylate and activate PKB directly (1–3), the PI3-K in- 3C). In F/B cells, in contrast to the ϳ4-fold activation of PDE3B, hibitor wortmannin, as expected, blocked IGF-1-induced activa- IGF-1 increased PDE4 activity only ϳ2-fold, as in F/V cells (Fig. tion of PKB and phosphorylation/activation of PDE3B in F/B cells 3C). As shown in Fig. 4, in FDCP2 cells expressing constitutively (Fig. 3). The mutant PKB expressed in F/B* cells, however, is not active PKB (F/B* cells), in the absence of IGF-1, PKB activity activated intrinsically by alteration at the catalytic site, rather it was ϳ10-fold greater than that in F/V cells (Fig. 4D). PDE3B was contains an N-terminal myristoylation sequence that targets it to phosphorylated (Fig. 4A) and activated (ϳ3- to 4-fold) (Fig. 4C) membranes, where it is presumably continuously or constitutively without change in the amount of immunoreactive PDE3B (Fig. activated in response to basal production of phosphoinositides with 4B). In F/B* cells, IGF-1 had little additional effect on activation activation of PDK isoforms or to a specific pool of active PI3-K. of PKB (see Fig. 1) or on phosphorylation/activation of PDE3B In F/B* cells (Fig. 4), wortmannin could inhibit constitutively ac- (data not shown, Table I). tivated PKB by blocking production of phosphoinositides involved As also shown in Fig. 3, wortmannin, in a concentration-depen- in PKB association with membranes and/or in the activation of dent manner, blocked the effects of IGF-1 on PKB (Fig. 3D), phos- specific PDK isoforms. Whether activated PKB contributes to phorylation/activation of PDE3B (Fig. 3, A and C), and activation feedback activation of PI3-K or sensitization of PDK to phosphoi- of PDE4 (Fig. 3C). As shown in Fig. 4, in F/B* cells, wortmannin, nositides is not known, but other workers have also reported that in a concentration-dependent manner, almost completely blocked wortmannin or LY294002 inhibits constitutively activated PKB ϳ (IC50, 25 nM) the elevated phosphorylation/activation of PDE3B (activated by targeting to membranes) in 3T3-L1 adipocytes (38) (Fig. 4, A and C) and PKB activity (Fig. 4D). or HEK293 cells (37). In nontransfected FDCP2 cells, wortmannin also blocked the Taken together, our previously reported data (26) and that pre- activation of PDE3B and PDE4 by IL-4 (26). Beyond PI3-K, how- sented in this report support the idea that in FDCP2 cells, IGF-1 ever, IL-4-effects on PDE3B and PDE4 diverged. IL-4-induced activates PDE3B and PDE4 via PI3-K-dependent signals. Down- activation of PDE3B was apparently independent of MAP kinase stream of PI3-K, PDE3B is phosphorylated/activated by activated and p70S6 kinase and not blocked by PD98059 (MEK-1 inhibitor) PKB, whereas PDE4 is activated via MEK/MAP kinase signals. or rapamycin (p70S6 kinase inhibitor). Activation of PDE4 by IL-4 was, however, dependent on MAP kinase and blocked by PD98059 (26). In FDCP2 cells expressing wt MEK, IL-3 activated Effects of PKB on phosphorylation/activation of PDE3B in vitro PDE4, not PDE3B; in FDCP2 cells expressing constitutively ac- Because effects of IGF-1 and wortmannin on phosphorylation/ac- tivated MEK, PDE4, not PDE3B, activity was increased in the tivation of PD3B in F/B or F/B* cells do not prove that PDE3B is absence of IL-3 (26). a direct substrate for PKB, we tested the ability of PKB to directly The Journal of Immunology 4683 Downloaded from http://www.jimmunol.org/

FIGURE 3. Effects of wortmannin on IGF-1-induced PDE3B phosphor- ylation, and PDE and PKB activities in F/V and F/B cells. FDCP2 cells, transfected with vector alone (F/V) or wt PKB (F/B), were incubated with- out or with the indicated concentrations of wortmannin for1hinRPMI ␮ 1640 medium containing 300 MKH2PO4 and TSB, and then with or 32 ␮ by guest on September 25, 2021 without added PO4 (40 Ci/ml) for 90 min before addition of the indi- FIGURE 4. Effects of wortmannin on PDE3B phosphorylation, and ϳ cated concentrations of IGF-1 for 10 min. A, Samples ( 2.5 mg protein) of PDE and PKB activities in transfected F/V and F/B* cells. FDCP2 cells, 32 lysates of cells incubated with PO4 were immunoprecipitated with anti- transfected with vector alone (F/V) or constitutively active PKB (F/B*), PDE3B-NT Ab and subjected to SDS-PAGE, and PDE3B bands were vi- were incubated without or with the indicated concentrations of wortmannin sualized by scanning the dried gel on a PhosphorImager (Molecular Dy- for1hinRPMI 1640 medium containing 300 ␮MKHPO and TSB, and ϳ 32 2 4 namics). Lysates ( 2.5 mg protein) of cells incubated without PO4 were 32 ␮ then with or without added PO4 (40 Ci/ml) for 90 min at 37°C. Samples immunoprecipitated and immunoblotted with anti-PDE3B Ab (B), or im- were prepared as described in the legend to Fig. 3. A, Immunoprecipitated munoprecipitated with anti-PKB-CT Ab (D) for assay of PKB activity by 32P-labeled PDE3B. B, PDE3B immunoprecipitated and immunoblotted Ϯ ϭ phosphorylation of Crosstide (mean cpm SEM, n 3). C, Samples of with anti-PDE3B-NT Ab. C, Total PDE (Ⅺ) and PDE3 (u) and PDE4 (f) 32 Ⅺ lysates of cells incubated without PO4 were assayed for total PDE ( ) activities (pmol/min/mg, mean Ϯ SEM (n ϭ 3)). D, PKB activity (mean u f Ϯ ϭ and PDE3 ( ) and PDE4 ( ) activities (mean SEM, n 3). cpm Ϯ SEM, n ϭ 3).

phosphorylation or removal of the N-terminal portion of the mol- phosphorylate and activate recombinant mouse (M)PDE3B. Pre- ecule. Taken together, these data suggest that PKB not only has a vious experiments have demonstrated that native RPDE3B was role as an upstream regulator, but also directly acts on PDE3B and phosphorylated on serine 302 in intact rat adipocytes (analogous to could thereby be an important determinant in control of intracel- serine 296 in MPDE3B) incubated with insulin (16), and that lular cyclic nucleotide concentrations. RPDE3B was also phosphorylated in vitro by partially purified rat adipocyte PKB (17) or rPKB (18). Available evidence suggests Effects of PDE inhibitors and cAMP on BAD phosphorylation that phosphorylation is not required to maintain, but does increase, and thymidine incorporation in F/V and F/B* cells basal PDE activity. As shown in Fig. 5, activated PKB, in a time- Activated PKB has been reported to increase cell proliferation/ and concentration-dependent manner, phosphorylated and in- survival (9, 11, 40), and to activate PDE3 and trigger meiosis in creased the activity of wt rMPDE3B synthesized in Sf-9 insect Xenopus oocytes (8). PKB-catalyzed phosphorylation of BAD may cells. As shown in Fig. 6, activated PKB did not, however, phos- be involved in the antiapoptotic effects of IL-3 (10). As shown in phorylate or increase the activity of M3B⌬604, a truncated Fig. 7A,in32P-labeled F/V and F/B cells, but not in F/BcϪ cells, rMPDE3B from which the N-terminal 604 aa (including putative IGF-1 increased phosphorylation of BAD, which was blocked by phosphorylation sites for PKB and PKA (16, 18)) were removed. incubation of F/B cells with 8-Br-cAMP or the PDE3 inhibitor The higher sp. act. of M3B⌬604 compared with that of wt cilostamide (Fig. 7B). Rolipram, a specific PDE4 inhibitor, was MPDE3B may be related to higher levels of expression or accu- much less effective in inhibiting IGF-1-stimulated phosphorylation mulation of a larger proportion of active M3B⌬604 molecules. It of BAD (Fig. 7B). These agents did not block IGF-1-induced ac- is also possible that the N-terminal portion of PDE3B contains an tivation of PKB (assessed by phosphorylation of Crosstide peptide autoinhibitory domain, the constraints of which are released by by immunoprecipitated PKB) (data not shown). 4684 PDE3B IS A TARGET AND EFFECTOR OF PKB ACTION

FIGURE 5. PKB-induced phosphorylation and acti- vation of wt rPDE3B in vitro. Recombinant wt PDE3B (ϳ10 ␮g SF-9 cell lysate protein) was incubated at 30°C with (Ⅺ) the indicated concentrations of com- mercially available (Upstate Biotechnology), activated PKB (0.3 ␮g/␮l) for 15 min (A) or for the indicated times with (Ⅺ) or without (ⅷ) activated (Upstate Bio- technology) PKB (ϳ1 ␮g) (B). The reaction mixtures (with and without [32P]ATP) contained 50 ␮M ATP, 5 ␮M PKA inhibitor, and 5 ␮M okadaic acid (A and B). Reactions (without [32P]ATP) were terminated by di- lution with PDE assay buffer and immediately assayed for PDE activity. Data are mean Ϯ SEM of three ex- periments, assayed in duplicate. To assess phosphory- lation of PDE3B (middle panels), reaction mixtures were boiled immediately in Laemmli buffer, and ana- lyzed by PhosphorImager after SDS-PAGE. Portions of reaction mixtures (without [32P]ATP) were immu- noblotted with anti-PDE3B Ab (bottom panels). Downloaded from

As shown in Fig. 8, in complete medium with IL-3, [3H]thymi- comparable with that in F/V cells grown in complete medium. dine incorporation was ϳ50% higher in F/B* cells than F/V cells. Whereas F/B* cells were able to proliferate and survive in the In the absence of IL-3, thymidine incorporation by F/B* cells was absence of IL-3, F/V cells were IL-3 dependent (as previously reported for nontransfected FDCP2 cells (25)). Increases in intracellular cAMP, brought about by agents that http://www.jimmunol.org/ activate adenylyl cyclase or by cAMP analogues that activate PKA, have been reported to induce apoptosis and/or inhibit pro- liferation in some cells, including lymphoid cells (41–44), granu- losa cells (45), tumor cells (46–48), and cultured smooth muscle cells (49). PDE inhibitors, by presumably increasing cAMP, have also been reported to inhibit proliferation/induce apoptosis of lym- phoid cells (50–53), cultured vascular smooth muscle cells (54), and tumor cells (55). As shown in Fig. 8, the PDE3 inhibitors by guest on September 25, 2021

FIGURE 7. BAD phosphorylation in PKB-transfected cells. Left, Ef- fects of IGF-1 in F/V, F/B, and F/BcϪ cells. Right, Effects of cilostamide, rolipram, PGE, and cAMP on IGF-1-induced BAD phosphorylation in F/B FIGURE 6. Effects of PKB on phosphorylation and activation of wt cells. FDCP2 cells, transfected with empty vector (F/V), kinase-inactive rPDE3B and a truncated PDE3B mutant in vitro. Recombinant wt PDE3B and (F/BcϪ), or wt (F/B) PKB, were incubated at 37°C in RPMI 1640 medium ⌬ ␮ PDE3B- 604 (a rPDE3B from which the first 604 aa were deleted) were with 300 MKH2PO4 and TSB, then with (A and B) or without (C, D, E, ϳ ␮ 32 ␮ incubated ( 10 g SF-9 cell lysate protein) at 30°C for 15 min with or without and F) added PO4 (100 Ci/ml) for 90–100 min, then as indicated for 20 20 ␮l of activated PKB (immunoprecipitated from lysates of F/V, F/B, and min with or without the PDE3 inhibitor cilostamide (10 ␮M), PDE4 in- Ϫ ␮ ␮ F/B cells after activation by incubation of cells for 10 min in the presence of hibitor rolipram (50 M), adenylate cyclase activator PGE1 (10 M), and 10 nM IGF-1) with or without [32P]ATP in reaction buffer containing 50 ␮M 8-Br-cAMP (500 ␮M), and finally for 10 min with or without 10 nM ATP, 5 ␮M PKA inhibitor, and 5 ␮M okadaic acid. To assess PDE3B phos- IGF-1. DMSO or ethanol at final concentrations of 0.1% or less was added phorylation (A), reaction mixtures were boiled immediately in Laemmli buffer, to appropriate wells as vehicle controls. A and B, BAD-32P was immuno- and analyzed by PhosphorImager analysis after SDS-PAGE. Reactions (with- precipitated with anti-BAD-NT Ab, and dried gels were scanned on a Phos- out [32P]ATP) were terminated by dilution with PDE assay buffer and imme- phorImager. C and D, Immunoprecipitates from cells incubated without 32P diately assayed for PDE activity (B). Data are mean Ϯ SEM of three exper- were prepared and immunoblotted with anti-BAD-NT Ab. E and F, Pro- iments, assayed in duplicate. Portions of reaction mixtures were also teins (ϳ30 ␮g) in lysates of control and PKB-transfected cells were sep- immunoblotted with anti-PKB-CT Ab (C), or anti-PDE3B-CT Ab (34) (D). arated by SDS-PAGE and immunoblotted with anti-PKB-CT Ab. The Journal of Immunology 4685

FIGURE 8. Thymidine incorporation into DNA in F/V and F/B* cells. FDCP2 cells transfected with vec- tor alone (F/V) (E, F), or constitutively active PKB (F/B*) (Ⅺ, f, ‚, Œ) were grown as described in Ma- terials and Methods without (‚, Œ) or with (E, F, Ⅺ, f) 5% WEHI (containing IL-3) and the indicated con- centrations of 8-Br-cAMP, PGE1, or PDE inhibitors (cilostamide, trequinsin, and rolipram) for 48 h. [3H]Thymidine was then added, and incubation was continued for 2 h before cells were harvested and DNA was collected on glass microfiber filters for radioassay, Ϯ as described in Materials and Methods. Data (mean Downloaded from SEM of values from triplicate determinations) are rep- resentative of two or three separate experiments. http://www.jimmunol.org/

trequinsin (A) and cilostamide (B) inhibited, in a concentration- ylation/activation of PDE3B, presumably resulting in a decrease in dependent manner, the enhanced thymidine incorporation in F/B* cAMP, could block some of these inhibitory effects of cAMP and cells grown in complete medium with IL-3, with little or no effect thus enhance downstream actions of PKB. Another signaling ki- in F/B* cells grown without IL-3. Rolipram (Fig. 8B), a specific nase, p70S6 kinase, is also apparently activated in cells expressing by guest on September 25, 2021 PDE4 inhibitor, was less effective than the PDE3 inhibitors. Al- constitutively activated PKB ((38), this study) and could also func- though PGE1 (which activates adenylyl cyclase) (Fig. 8D) was tion as an effector for PKB. There is, however, no direct evidence rather ineffective in inhibiting thymidine incorporation, higher to suggest that in intact cells p70S6 kinase is a substrate for PKB. concentrations of 8-Br-cAMP did inhibit thymidine incorporation cAMP, however, was also reported to inhibit PI3-K-induced acti- by F/V and F/B* cells in complete medium, but had little effect in vation of p70S6 kinase in IL-2-responsive cells (63). Specific cell F/B* cells grown without IL-3 (Fig. 8C). As shown in Fig. 9, however, at concentrations that were not very effective alone, com- binations of PGE1 and cilostamide or cilostamide and rolipram had additive or synergistic inhibitory effects on thymidine incorpora- tion in F/B cells and F/B* cells grown in complete medium, and especially in F/B* cells grown without IL-3. The combination of

PGE1 and rolipram was not as effective as PGE1 and cilostamide or cilostamide and rolipram. These results suggest that, although increases in cAMP brought about by either activation of adenylyl cyclase/PDE3 inhibition or inhibition of both PDE3 and PDE4 can markedly inhibit thymidine incorporation (Fig. 9), the role of PDE3 appears predominant. The detailed mechanism(s) by which cAMP inhibits cell prolif- eration/survival has not been completely identified and may in- volve inhibition of autocrine growth factors and cytokines such as IL-2 (50, 56) or direct interruption of mitogenic signaling path- ways. Sustained elevation of intracellular cAMP can induce arrest during G or G phases of the cell cycle (42, 46, 47). cAMP was FIGURE 9. Thymidine incorporation into DNA in F/V and F/B* cells. 1 2 Ⅺ u f reported to inhibit Ras/Raf-1 activation of ERK signaling as well F/V ( ) and F/B* ( , ) cells were grown as described in Materials and Methods in the absence (f) or presence (u, Ⅺ) of 5% WEHI-conditioned as cyclin kinase 4, c-Jun N-terminal kinase, c-Jun, NF-␬B, and Stat medium (containing IL-3) and the indicated concentrations of PGE1, cilos- 1 expression (56–61). Activation of the type I PKA by cAMP tamide, or rolipram, alone or in combination for 48 h. [3H]Thymidine was analogues inhibits proliferation of lymphocytes (44) and meiotic added and incubation was continued for 2 h before cells were harvested and maturation of mouse oocytes (62). Although it is highly likely that DNA collected on glass microfiber filters. Data (mean Ϯ SEM of values other downstream targets of PKB, e.g., BAD, directly participate from triplicate determinations) are representative of three separate in the antiapoptotic effects of PKB (10), PKB-catalyzed phosphor- experiments. 4686 PDE3B IS A TARGET AND EFFECTOR OF PKB ACTION context may be important in the function of PDE3B and cAMP in the action of PKB on cell proliferation/survival, since, in some cells, cAMP actually promotes growth (48, 64) and prevents or delays apoptosis (65). The proapoptotic protein BAD, a member of the Bcl-2 , promotes apoptosis by heterodimerization with the survival factors Bcl-2 or Bcl-xL. Bcl or Bcl-xL suppresses apoptosis, at least in part, by preventing release of cytochrome c from mito- chondria, and thus blocking activation of caspase proteases (66). Survival factors, including IGF-1, IL-3, nerve growth factor, and other cytokines, induce phosphorylation of BAD on critical serine FIGURE 10. Postulated role for PKB-induced activation of PDE3 in effects of growth factors such as insulin and IGF-1 on several cAMP- residues, leading to its interaction with and sequestration by 14-3-3 mediated processes. proteins, its dissociation from the BAD/Bcl complex, and subse- quent translocation from mitochondria to the cytosol; free Bcl pro- teins act as suppressors of apoptosis (66). Although it is clear that PKB-induced phosphorylation of BAD dogenous MPDE3B in 3T3L1 adipocytes, they did report that can play an important role in mechanisms that regulate cell sur- serine 276 (within an RXRXXS motif) in rMPDE3B is phosphor- vival/proliferation (10, 67), in some cells other signaling pathways ylated by activated PKB. Current studies in our laboratories are involving Raf-1 and PKA regulate phosphorylation of BAD (68), attempting to reconcile these apparent differences and identify the Downloaded from and PKB-independent pathways involving MEK and MAP kinases site(s) involved in phosphorylation/activation of PDE3B by PKB suppress apoptosis (68–70). Furthermore, in lymphocytes, activa- in vitro and intact cells. 2ϩ tion of the TCR can induce apoptosis via Ca -induced activation Insulin, IGF-1, or IL-4, each of which utilizes IRS proteins to of the (PP2b) (71), resulting in dephos- initiate at least some of its receptor signaling cascades, activates phorylation of BAD, its heterodimerization with Bcl proteins, and PDE3 in adipocytes (16, 17, 21), FDCP2 cells, pancreatic ␤ cells initiation of apoptosis (72). (77), hepatocytes (78), and Xenopus oocytes (8, 79, 80). In these http://www.jimmunol.org/ Recently, Minishall et al. (73) demonstrated that IGF-1 and IL-4 cells, some effects of the polypeptides are counterregulatory to increased expression of Bcl-2 and survival of FDCP2 cells via those of cAMP, which increases (19, 20, 81), inhibits cell PI3-K-dependent pathways. In that study, effects of IGF-1 on PKB proliferation/survival (41–55), stimulates insulin secretion (77) and BAD phosphorylation, or of cAMP on the antiapoptotic ac- and glycogenolysis (82), and blocks meiosis (8), respectively. It is, tions of IGF-1 were not assessed. Our results indicate that in therefore, tempting to speculate that in these cells, activation of FDCP2 cells, IGF-1 activates PDE3 and PDE4 via PI3-K-depen- PDE3, which would presumably reduce cAMP and PKA, may play dent pathways (29). Downstream of PI3-K, PDE3 is activated by an important role in the counterregulatory effects of the polypep- PKB-dependent and PDE4 by MEK/MAP kinase-dependent (29) tides, i.e., inhibiting lipolysis (16, 19–21), enhancing cell prolif- signals. In this context, our findings are consistent with the idea eration, inhibiting insulin secretion (77) and glycogenolysis (82), by guest on September 25, 2021 that in FDCP2 cells, both the extent and duration of increased and stimulating meiosis (8, 79, 80), respectively (Fig. 10). From a intracellular cAMP may be important in regulation of BAD phos- broader perspective, these and other studies are consistent with the phorylation and thymidine incorporation, and that PDEs (especial- idea that PDEs comprise a complex group of structurally related ly PDE3B) can influence these parameters and, consequently, cell and highly regulated enzymes that are critical, if not essential, in survival. It is possible that in FDCP2 cells, increased cAMP and influencing specificity, compartmentation, and overall regulation activation of PKA could directly or indirectly block association of of cyclic nucleotide-mediated processes (16, 83–85). 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