The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10–40 μm dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5–20 μm inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

The differentiation-inducing factor-1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one; Fig. 1] is a signal molecule that induces stalk cell differentiation in Dictyostelium discoideum(1, 2). DIF-3 (Fig. 1), a dechlorinated form of DIF-1, is the first metabolite of DIF-1, whose stalk-inducing activity is very low in D. discoideum(2, 3); on the other hand, 2-methoxy isomer of DIF-1 (2-MIDIF-1) and 6-methoxy isomer of DIF-3 (6-MIDIF-3; Fig. 1) are the artificially synthesized isomers of DIF-1 and DIF-3, respectively.

Recently, it has been shown that DIF-1 and DIF-3 exhibit antiproliferative activities and occasionally induce cell differentiation in mammalian cells (4, 5, 6, 7, 8, 9, 10, 11, 12). Some results obtained by our research group, namely, that DIF-1 increases [Ca2+]i in some tumor cells (6, 7, 8, 9), activates Akt/protein kinase B human leukemia K562 cells (9), inactivates signal transducer and activator of transcription 3 (STAT3) in gastric cancer cells (11), and down-regulates G1 cyclins in vascular smooth muscle cells (10), are significant in the elucidation of the molecular mechanism of the actions of DIF-1 in mammalian cells. Moreover, we have found that DIF-1 may block a decrease in the intracellular cyclic AMP concentration ([cAMP]i) induced by progesterone and thereby inhibit progesterone-induced oocyte maturation in Xenopus laevis(13). Yet, the precise signaling system of DIF-1 and, especially, the target molecule(s) of DIF-1 are unknown in Dictyostelium, Xenopus, and mammalian cells.

In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, and we show here that pharmacological concentrations of DIF-1 inhibit the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro. Because DIF-1 did not significantly affect the activity of some other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1 in mammalian cells.

Reagents and Enzymes.

DIF-1 and its analogs were synthesized by Toyama Chemical Co. Ltd. (Toyama, Japan) following the method of Masento et al.(14). Bovine brain calcineurin, CaM, PDE1, p-nitrophenylphosphate, and bis-p-nitrophenylphosphate were obtained from Sigma (St. Louis, MO), snake venom PDE (svPDE) was from the Worthington Biochemical Corporation (Lakewood, NJ), and calf-intestinal alkaline phosphatase (AP) was from New England BioLabs (Beverly, MA). Smooth-muscle myosin and myosin light-chain kinase were purified from chicken gizzard as described previously (15, 16). The definitions of units (U) for enzymatic activities used in this study are in accordance with the manufacturer’s descriptions.

Assay for Cell Growth.

The human leukemia K562 cells were maintained at 37°C (5% CO2) in a growth medium (RPMI 1640 with 10% fetal bovine serum; designated RPMI), and cell growth was assessed as described previously (8). Briefly, cells were incubated in a multi(12)-well plate, each well containing 1 ml of RPMI (5 × 104 cells/ml) in the presence or absence of DIF-analogs or inhibitors for CaM or PDEs. On day 3, a 1:20 volume (50 μl) of Alamar Blue (cell number indicator) was added to each well, and after a 1–2-h incubation at 37°C (5% CO2), 150 μl of each of the sample solutions were transferred into a 96-well plate, and absorbance at 570 nm (reference at 595 nm) was measured with a microplate reader (Bio-Rad; Model 550). A cell number was given as % of the control absorbance.

Assay for [cAMP]i.

Cells were suspended (2 × 106 cells/ml) in an assay buffer [20 mm HEPES-NaOH (pH 7.4), 137.5 mm NaCl, 5 mm KCl, 1.5 mm CaCl2, 0.8 mm MgCl2, 5.5 mm glucose, 0.6 mm NaHCO3, and 0.1% (w/v) BSA]. One ml of the cell suspension in a tube was incubated at room temperature with or without 10–40 μm DIF-1 for the indicated minutes (Fig. 2), and the cells were collected by quick centrifugation (3500 rpm, 30 s), resuspended in 100 μl of 0.2 n HCl, and destroyed by mild sonication. The samples were neutralized and assayed for cAMP content as described previously (17).

Assay for PDE1 Activity.

Bovine PDE1 (0.75 mU), CaM (10 mU), and CaCl2 (0.2 mm) were incubated at 30°C for 10 min in 0.3 ml of a reaction buffer {50 mm HEPES-NaOH (pH 7.5), 0.1 mm EGTA, 8.3 mm MgCl2, 0.5 μm [3H]cAMP (18,000 cpm)} containing DIF-analogs or inhibitors. In studies measuring PDE1 activity in the absence of CaM, PDE1 (3 mU) was incubated at 30°C for 15 min in a reaction buffer containing DIF-analogs or inhibitors. PDE1 activity was assayed by a modification of a previously described procedure (18).

Assay for PDE3A, PDE3B, and PDE8A.

Baculoviruses of the human full-length PDE3A, human PDE3A NH2-terminal deletion mutant (amino acids 511-1141; Ref. 19), human full-length PDE3B (20), and rat PDE3B NH2-terminal deletion mutant (amino acids 577-1108; Ref. 21) were gifts of Dr. V. C. Manganiello (NIH, Bethesda, MD). Mouse PDE8A was prepared as follows. Total RNA was isolated from mouse osteoblast MC3T3-E1 cells using the RNeasy mini kit (Qiagen, Hilden, Germany). A specific oligonucleotide primer set was synthesized: 5′-CCTAAATGTCTGCCTCGTTTGCTAGTG-3′ and 5′-GTGCCGCCGCCGCCAGTATGGGCTGCGCCCCG-3′; and reverse transcription-PCR was carried out using the Qiagen OneStep RT-PCR kit (Qiagen). The purified PCR product was introduced into the plasmid pCR II-TOPO vector (Invitrogen, Carlsbad, CA) and was verified by DNA sequencing. The cDNA for mouse PDE8A was subcloned into the BamHI/XbaI sites of the pVL1393. Transfer of the cDNA from pVL1393 to the Autographa California nuclear polyhedrosis virus was accomplished using the BaculoGold Transfection kit (PharMingen, San Diego, CA). High-titer recombinant viral stocks encoding PDE8A were obtained and were used for the subsequent infection of Sf9 insect cells (PharMingen).

The membrane-integrated (full-length) and soluble (truncated) forms of PDE3A and PDE3B and the soluble (full-length) PDE8A were expressed in Sf9 cells as described previously (22). Sf9 cells were maintained, propagated at 27°C in the TNM-FH Insect Medium (PharMingen), and were collected and sonicated in 1 ml of a homogenization buffer [100 mm Tris-HCl (pH 7.4) and 5 mm MgSO4]. The homogenized cell samples containing full-length PDE3A or PDE3B were used for the assay for PDE activity. And the homogenized cells containing the truncated forms of PDE3A or PDE3B or the full-length PDE8A were centrifuged (100,000 × g, 60 min, 4°C) to obtain soluble fractions, which were also used for the assay for PDE activity.

Samples were incubated at 30°C for 10 min in 0.3 ml of a reaction buffer {50 mm HEPES-NaOH (pH 7.4), 0.1 mm EGTA, 8.3 mm MgCl2, 0.1 μm [3H]cAMP (18,000 cpm)} in the presence of DIF-1 or inhibitors. PDE activity was assayed as described previously (18).

Assay for the Other Enzyme Activities.

Three units each of bovine calcineurin (∼ 0.1 μm) and CaM (∼ 0.2 μm) were incubated at 30°C for 60 min in 200 μl of a reaction buffer in the presence or absence of various concentrations of DIF-1, and calcineurin activity was assayed as described previously (23).

Chicken gizzard myosin (3.7 μm) was phosphorylated with 0.086 μm myosin light-chain kinase and 0.3 μm CaM in a reaction buffer (23) in the presence or absence of 10–20 μm DIF-1 or 10 μm calmidazolium (CZM) at 25°C for 20 min. After terminating the reaction by adding an equal volume of a sample buffer [6 m urea, 14 mm 2-mercaptoethanol, and 50 mm Tris-HCl (pH 6.8)], samples were analyzed with urea-glycerol PAGE as described previously (24).

svPDE (0.1 unit) was incubated at 37°C for 15 min in 200 μl of an assay buffer [138 mm Tris-HCl (pH 8.7), 9 mm bis-p-nitrophenylphosphate], and the reaction was stopped by adding 800 μl of 1 m Na2CO3. svPDE activity was quantified by measuring the A410 nm of the reaction mixtures.

AP (26 mU) was incubated at 37°C for 5 min in 200 μl of an assay buffer [0.4 m glycine-NaOH (pH 10.5), 1 mm MgCl2, 0.1 mm ZnCl2, and 6 mmp-nitrophenylphosphate] in the presence of various concentrations of DIF-1, and AP activity was assessed as described previously (23).

We first examined whether DIF-1 would affect [cAMP]i in K562 leukemia cells (Fig. 2). DIF-1 at 10–40 μm slightly increased [cAMP]i (Fig. 2,A) in a dose-dependent manner (Fig. 2,B), inducing growth arrest (Fig. 2,C; Refs. 4, 8). Although the physiological significance of the slight increase in [cAMP]i was not known, this finding seemed to provide a hint for the identification of the pharmacological target(s) of DIF-1 in mammalian cells; it was likely that DIF-1 might affect cAMP production and/or degradation. Moreover, because some inhibitors for CaM and cyclic nucleotide PDEs showed antiproliferative activity in K562 cells (Fig. 2,C), we examined the effects of DIF-1 on some PDEs and CaM-dependent enzymes in vitro (Figs. 3,4,5,6). It was then found that DIF-1 at pharmacological concentrations inhibited the activity of CaM-dependent cyclic nucleotide PDE1 in a dose-dependent manner (Fig. 3 A).

To assess whether DIF-1 binds to PDE1 or CaM, we examined the effect of DIF-1 on PDE1 in the absence of CaM (Fig. 3,B). W-7 did not inhibit PDE1 activity in the absence of CaM, whereas CZM, which can directly bind not only to CaM but also to PDE1 (25), inhibited PDE1 activity (Fig. 3,B). In the absence of CaM, DIF-1 at the pharmacological concentration range inhibited PDE1 activity dose-dependently (Fig. 3,B), which suggested that PDE1 but not CaM should be the target of DIF-1. A comparative study using DIF-analogs showed that DIF-1 and DIF-3 (DIFs) were potent inhibitors for PDE1 both in the presence (Fig. 3,C) and in the absence (Fig. 3,D) of CaM, whereas the effects of 2-MIDIF-1 and 6-MIDIF-3 were weaker than those of DIFs (Fig. 3, C and D), suggesting that PDE1 inhibition by DIFs should be chemical structure-specific. Quite interestingly, kinetic analyses (Fig. 4) revealed that DIF-1 should act as a competitive inhibitor of PDE1 toward cAMP, and the Ki value was calculated to be 4.5–5 μm.

We then examined the effects of DIF-1 on some other cyclic nucleotide PDEs (Fig. 5). DIF-1 at up to 50 μm did not affect the activity of the cyclic-GMP-inhibited cAMP-specific PDEs, PDE3A, and PDE3B (Ref. 26; Fig. 5,A). However, because the original forms of PDE3A and PDE3B are membrane-integrated proteins and the crude cell lysates in the assay mixture might have interfered with the action of DIF-1, which is a lipophilic substance, we examined the effects of DIF-1 on soluble (truncated) forms of PDE3A and PDE3B in the absence of the debris of cell membranes (Fig. 5,B). Again, DIF-1 did not affect the activity (Fig. 5,B). On the other hand, DIF-1 at 50 μm reduced the activity of PDE8A, a cAMP-specific PDE (27), by ∼40% (Fig. 5,C), but the effect of DIF-1 on this enzyme was much weaker than that on PDE1 (Fig. 3 A). Thus, PDE8A inhibition by DIF-1 should be relatively nonspecific.

We further examined the effects of DIF-1 on two other CaM-dependent enzymes (calcineurin and myosin light-chain kinase), svPDE, and AP. DIF-1 at up to 10 μm did not inhibit calcineurin activity, and DIF-1 at 30 μm reduced the activity by ∼20% (Fig. 6,A), whereas myosin light-chain kinase activity was not affected by DIF-1 at 20 μm (Fig. 6 B). It was also found that DIF-1 at up to 30 μm did not affect svPDE or AP (data not shown). All of the results strongly suggest that PDE1 is a pharmacological and specific target of DIF-1.

As already mentioned, the target molecule(s) of DIF-1 had not been identified in either mammals or the original organism, Dictyostelium, despite efforts by researchers (28). Here, we have shown for the first time that PDE1 is a pharmacological and specific target of DIF-1 and DIF-3 (designated DIFs) in mammalian cells. It should be of importance to note here that some inhibitors for PDE1 are expected to have therapeutic potential in the treatment of cancer (29, 30), which agrees well with the fact that DIFs are potent antitumor agents (4, 5, 6, 7, 8, 9, 10, 11).

The inhibitory effect of CZM on PDE1 is slightly weaker than that of DIF-1 (Fig. 3,B), whereas CZM is the most potent inhibitor of cell growth among the drugs tested in Fig. 2,C, probably because CZM is a broad inhibitor for CaM-dependent enzymes. 3-isobutyl-1-methylxanthine (IBMX) and 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MIBMX) should be good inhibitors for PDEs and PDE1, respectively, but with the inhibitors at relatively higher concentrations, cell growth can be suppressed (Fig. 2 C). The therapeutic advantage, if any, of DIFs as compared with those of other PDE inhibitors is not known at present. However, in general, because membrane-permeable and more specific inhibitors of PDE1 should be good tools for both experimental and therapeutic uses, DIFs and/or their derivatives may be such drugs.

Although how much DIFs can penetrate the membrane and inhibit intracellular PDE1 is unknown, the concentration range of DIFs for growth inhibition (Fig. 2,C) roughly corresponds to the concentration range of DIFs for PDE1 inhibition in vitro (Fig. 3, A–D). However, because the correlation between growth inhibition (Fig. 2,C) and PDE1 inhibition (Fig. 3, C and D) by the DIF-analogs was not perfect, it is still possible that there may be some other target(s) of DIFs.

As a next step, we will need to further verify whether the known actions of DIFs in mammalian cells are exerted via PDE1 inhibition by DIFs and whether DIFs affect some other PDEs and/or enzymes relating to cAMP. In addition, it should be assessed in the future whether this is the case in Dictyostelium. At any rate, the present information that DIF-1 directly inhibits PDE1 in vitro by competing with cAMP should provide a new insight into, and a hint for the identification of, some other target(s), if any, of DIFs.

Grant support: Supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (Y. Kubohara).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: K. Shimizu and T. Murata contributed equally to this work.

Requests for reprints: Kubohara, Biosignal Research Center, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512, Japan. Phone: 81-27-220-8866; Fax: 81-27-220-8897; E-mail: [email protected]

Fig. 1.

Chemical structure of DIF-analogs. DIF-1, differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one; DIF-3, differentiation-inducing factor-3 [1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one]; 2-MIDIF-1, 2-methoxy isomer of DIF-1; 6-MIDIF-3, 6-methoxy isomer of DIF-3.

Fig. 1.

Chemical structure of DIF-analogs. DIF-1, differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one; DIF-3, differentiation-inducing factor-3 [1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one]; 2-MIDIF-1, 2-methoxy isomer of DIF-1; 6-MIDIF-3, 6-methoxy isomer of DIF-3.

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Fig. 2.

Effects of differentiation-inducing factor-1 (DIF-1) on intracellular cyclic AMP ([cAMP]i) and cell growth in K562 cells. A, K562 cells were incubated for the indicated times with 20 μm DIF-1, and cAMP content was assessed. Values are the mean ± SD of three independent experiments (n = 3). ∗∗, P < 0.03 versus ∗control (by ANOVA, post hoc Fisher’s protected least significant difference). B, K562 cells were incubated for 5 min with 10–40 μm DIF-1, and cAMP content was assessed. Values are the mean ± one-half of the range of two independent experiments (n = 2). DIF-1 increased [cAMP]i in a dose-dependent manner. ∗∗, P < 0.05 versus ∗control; ∗∗∗, P < 0.005 versus ∗control. C, K562 cells were incubated for 3 days with the indicated drugs, and the cell number was assessed. Drugs used are the DIF-analogs, DIF-1, DIF-3, 2-methoxy isomer of DIF-1 (2-MIDIF-1), and 6-methoxy isomer of DIF-3 (6-MIDIF-3); the calmodulin inhibitors, calmidazolium (CZM) and W-7; the PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MIBMX); and the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Values are the mean ± SD of three independent experiments (n = 3).

Fig. 2.

Effects of differentiation-inducing factor-1 (DIF-1) on intracellular cyclic AMP ([cAMP]i) and cell growth in K562 cells. A, K562 cells were incubated for the indicated times with 20 μm DIF-1, and cAMP content was assessed. Values are the mean ± SD of three independent experiments (n = 3). ∗∗, P < 0.03 versus ∗control (by ANOVA, post hoc Fisher’s protected least significant difference). B, K562 cells were incubated for 5 min with 10–40 μm DIF-1, and cAMP content was assessed. Values are the mean ± one-half of the range of two independent experiments (n = 2). DIF-1 increased [cAMP]i in a dose-dependent manner. ∗∗, P < 0.05 versus ∗control; ∗∗∗, P < 0.005 versus ∗control. C, K562 cells were incubated for 3 days with the indicated drugs, and the cell number was assessed. Drugs used are the DIF-analogs, DIF-1, DIF-3, 2-methoxy isomer of DIF-1 (2-MIDIF-1), and 6-methoxy isomer of DIF-3 (6-MIDIF-3); the calmodulin inhibitors, calmidazolium (CZM) and W-7; the PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MIBMX); and the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Values are the mean ± SD of three independent experiments (n = 3).

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Fig. 3.

Effects of differentiation-inducing factor-1 (DIF-1) and its analogs on phosphodiesterase (PDE1) activity. A, bovine PDE1 (5 mU/tube) was incubated in vitro in an assay buffer with various concentrations of DIF-1 (•) or W-7 (○) in the presence (○, •, + CaM) or absence (▪, − CaM) of bovine calmodulin (CaM; 10 units/tube) for 10 min, and the samples were assayed for enzymatic activity. Every sample contains 1% (v/v) ethanol as a vehicle. Values are the mean ± one-half of the range of two independent experiments (n = 2). B, PDE1 (10 mU/tube) was incubated in an assay buffer with various concentrations of DIF-1(•), W-7(○), or calmidazolium (CZM; ▴) in the absence of CaM (− CaM); and the samples were assayed for enzymatic activity. Values are the mean ± one-half of the range of two independent experiments (n = 2). C, PDE1 (5 mU/tube) was incubated in an assay buffer with 5 μm of DIF-1, DIF-3, 2-methoxy isomer of DIF-1 [2-MIDIF-1 (2-MID-1)], or 6-methoxy isomer of DIF-3 [6-MIDIF-3 (6-MID-3)], or with 50 μm W-7, 10 μm CZM, or 1% ethanol (Vehicle) in the presence (+) or absence (-) of CaM (10 units/tube) for 10 min; and the samples were assayed for enzymatic activity. Values are the mean ± SD of three independent experiments (n = 3). ∗, P < 0.001; ∗∗, P < 0.005. D, PDE1 (10 mU/tube) was incubated in an assay buffer with 5 μm of DIF-1, DIF-3, 2-MIDIF-1 (2-MID-1), or 6-MIDIF-3 (6-MID-3), or with 50 μm W-7, 10 μm CZM, or 1% ethanol (Vehicle) in the absence of CaM (− CaM) for 10 min; and the samples were assayed for enzymatic activity. Values are the mean ± SD of three independent experiments (n = 3). ∗, P < 0.001; ∗∗, P < 0.05.

Fig. 3.

Effects of differentiation-inducing factor-1 (DIF-1) and its analogs on phosphodiesterase (PDE1) activity. A, bovine PDE1 (5 mU/tube) was incubated in vitro in an assay buffer with various concentrations of DIF-1 (•) or W-7 (○) in the presence (○, •, + CaM) or absence (▪, − CaM) of bovine calmodulin (CaM; 10 units/tube) for 10 min, and the samples were assayed for enzymatic activity. Every sample contains 1% (v/v) ethanol as a vehicle. Values are the mean ± one-half of the range of two independent experiments (n = 2). B, PDE1 (10 mU/tube) was incubated in an assay buffer with various concentrations of DIF-1(•), W-7(○), or calmidazolium (CZM; ▴) in the absence of CaM (− CaM); and the samples were assayed for enzymatic activity. Values are the mean ± one-half of the range of two independent experiments (n = 2). C, PDE1 (5 mU/tube) was incubated in an assay buffer with 5 μm of DIF-1, DIF-3, 2-methoxy isomer of DIF-1 [2-MIDIF-1 (2-MID-1)], or 6-methoxy isomer of DIF-3 [6-MIDIF-3 (6-MID-3)], or with 50 μm W-7, 10 μm CZM, or 1% ethanol (Vehicle) in the presence (+) or absence (-) of CaM (10 units/tube) for 10 min; and the samples were assayed for enzymatic activity. Values are the mean ± SD of three independent experiments (n = 3). ∗, P < 0.001; ∗∗, P < 0.005. D, PDE1 (10 mU/tube) was incubated in an assay buffer with 5 μm of DIF-1, DIF-3, 2-MIDIF-1 (2-MID-1), or 6-MIDIF-3 (6-MID-3), or with 50 μm W-7, 10 μm CZM, or 1% ethanol (Vehicle) in the absence of CaM (− CaM) for 10 min; and the samples were assayed for enzymatic activity. Values are the mean ± SD of three independent experiments (n = 3). ∗, P < 0.001; ∗∗, P < 0.05.

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Fig. 4.

Kinetic analysis of differentiation-inducing factor-1 (DIF-1)-induced inhibition of phosphodiesterase (PDE1) activity. PDE1 and cyclic AMP (cAMP) were incubated in an assay buffer with various concentrations (0.5–5 units) of calmodulin (CaM) in the presence or absence of DIF-1 (A), or PDE1 and CaM were incubated with various concentrations (0.2–1 μm) of cAMP in the presence or absence of DIF-1 (B), and the samples were assayed for enzymatic activity. The data were plotted as 1/v versus 1/[CaM] (A) or 1/[cAMP] (B), and the fittest lines were determined by linear regression analysis. Each result is a representative of three independent experiments. The results indicate that DIF-1 is a noncompetitive inhibitor toward CaM and a competitive inhibitor toward cAMP.

Fig. 4.

Kinetic analysis of differentiation-inducing factor-1 (DIF-1)-induced inhibition of phosphodiesterase (PDE1) activity. PDE1 and cyclic AMP (cAMP) were incubated in an assay buffer with various concentrations (0.5–5 units) of calmodulin (CaM) in the presence or absence of DIF-1 (A), or PDE1 and CaM were incubated with various concentrations (0.2–1 μm) of cAMP in the presence or absence of DIF-1 (B), and the samples were assayed for enzymatic activity. The data were plotted as 1/v versus 1/[CaM] (A) or 1/[cAMP] (B), and the fittest lines were determined by linear regression analysis. Each result is a representative of three independent experiments. The results indicate that DIF-1 is a noncompetitive inhibitor toward CaM and a competitive inhibitor toward cAMP.

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Fig. 5.

Effect of differentiation-inducing factor-1 (DIF-1) on the activity of PDE3A, PDE3B, and PDE8A. A, Sf9 cell lysates containing the original form (membrane-integrated form) of human PDE3A or rat PDE3B were incubated in an assay buffer in the presence of DIF-1 or cilostamide, and phosphodiesterase (PDE) activity was assessed. Values are the mean ± one-half of the range of two independent experiments (n = 2). B, cell lysates containing a soluble form of PDE3A or PDE3B were centrifuged, and the enzymatic activity in the supernatants was assessed in the presence of DIF-1 or cilostamide (PDE3 inhibitor). Values are the mean ± SD of three independent experiments (n = 3). C, cell lysates containing the original form (soluble form) of mouse PDE8A were centrifuged, and the enzymatic activity in the supernatant was assessed in the presence of DIF-1 or dipyridamole (PDE8 inhibitor). Values are the mean ± SD of three independent experiments (n = 3).

Fig. 5.

Effect of differentiation-inducing factor-1 (DIF-1) on the activity of PDE3A, PDE3B, and PDE8A. A, Sf9 cell lysates containing the original form (membrane-integrated form) of human PDE3A or rat PDE3B were incubated in an assay buffer in the presence of DIF-1 or cilostamide, and phosphodiesterase (PDE) activity was assessed. Values are the mean ± one-half of the range of two independent experiments (n = 2). B, cell lysates containing a soluble form of PDE3A or PDE3B were centrifuged, and the enzymatic activity in the supernatants was assessed in the presence of DIF-1 or cilostamide (PDE3 inhibitor). Values are the mean ± SD of three independent experiments (n = 3). C, cell lysates containing the original form (soluble form) of mouse PDE8A were centrifuged, and the enzymatic activity in the supernatant was assessed in the presence of DIF-1 or dipyridamole (PDE8 inhibitor). Values are the mean ± SD of three independent experiments (n = 3).

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Fig. 6.

Effect of differentiation-inducing factor-1 (DIF-1) on calcineurin and myosin light-chain kinase (MLCK). A, bovine calcineurin (CN) and calmodulin were incubated in an assay buffer with various concentrations of DIF-1 or calmidazolium (CZM) and assayed for CN activity. Values are the mean ± SD of three independent experiments (n = 3). B, chicken gizzard myosin, MLCK, and CaM were incubated in an assay buffer in the presence or absence of 10–20 μm DIF-1 or 10 μm CZM, and the samples were analyzed by urea-glycerol PAGE. Arrows, the positions of myosin light chain (Myosin Lc) and phosphorylated myosin light chain (P-Myosin Lc). A representative result of three independent experiments is shown.

Fig. 6.

Effect of differentiation-inducing factor-1 (DIF-1) on calcineurin and myosin light-chain kinase (MLCK). A, bovine calcineurin (CN) and calmodulin were incubated in an assay buffer with various concentrations of DIF-1 or calmidazolium (CZM) and assayed for CN activity. Values are the mean ± SD of three independent experiments (n = 3). B, chicken gizzard myosin, MLCK, and CaM were incubated in an assay buffer in the presence or absence of 10–20 μm DIF-1 or 10 μm CZM, and the samples were analyzed by urea-glycerol PAGE. Arrows, the positions of myosin light chain (Myosin Lc) and phosphorylated myosin light chain (P-Myosin Lc). A representative result of three independent experiments is shown.

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