The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by...

THE JUURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 12, Issue of March 25, pp. 9245-9252, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

The Short-term Activation of a Rolipram-sensitive, cAMP-specific Phosphodiesterase by Thyroid-stimulating Hormone in Thyroid FRTL-5 Cells Is Mediated by a CAMP-dependent Phosphorylation*

(Received for publication, October 8, 1993, and in revised form, December 14, 1993)

Claudio Settet, Saveria Iona, and Marco Conti From the Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford Medical Center,

” . Stanford, California 94305-531 7

To elucidate the mechanism causing the transient accumulation of intracellular CAMP in the FRTL-5 thyroid cell line, the short-term effect of thyroid-stimulating hormone (TSH) on phosphodiesterase (PDE) activity was studied. Together with an increase in CAMP levels, TSH produced a significant increase in total PDE activity as early as 3 min, with a maximal stimulation reached after 15 min. This short-term increase in PDE activity was dependent on the TSH concentration (EDSO = 4 x lo-” M TSH). Forskolin and dibutyryl CAMP produced an even larger stimulation than that produced by TSH, suggesting that the effect of TSH is mediated by CAMP. To determine the properties of the PDE forms activated by TSH, antibodies specific for the CAMP- PDEs were used to immunoprecipitate the PDEs present in control cells, and cells incubated for 15 min in the presence of 10 nM TSH. Comparison of the activity recovered in the immunoprecipitation pellets demonstrated that TSH produced more than a 2.5-fold increase in the CAMP-PDE form(s) recognized by this antibody. Conversely, the activity remaining in the supernatants was not affected by the TSH treatment. Most of the activity recovered in the immunoprecipitation pellets (90%) was inhibited by 10 p~ Rolipram, an inhibitor specific for the high affinity CAMP-PDEs. No TSH stimulation of the Rolipram-insensitive PDE activity could be observed under these conditions. Western blot analyses with two different CAMP-PDE specific antibodies showed that a 15-min stimulation with TSH induced the appearance of a new band with electrophoretic mobility slower than the polypeptide present in unstimulated cells. The appearance of this band did not require ongo- ing protein synthesis because it occurred in the presence of cycloheximide. Metabolic [32P]orthophosphate labeling of intact FRTL-5 cells indicated that the TSH treatment caused an increased 32P incorporation into a polypeptide that co-purified with the stimulated PDE activity and had an electrophoretic mobility identical to that of the CAMP-PDE. Okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A, elicited a potentiation of the TSH-stimulated PDE activity. The stimulation of a PDE with the same immunological properties and Rolipram sensitivity as the CAMP- PDE stimulated by TSH in the intact cells was reproduced, in a cell-free system, by incubating soluble extracts from FRTL-5 cells with the catalytic subunit of

Grant HD20788 and a grant from Institute Pasteur-Fondazione Cenci * This work was supported in part by National Institutes of Health

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

$ Supported by a fellowship grant from Stanford University.

CAMP-dependent protein kinase. These data provide evidence that TSH produces a rapid activation of a CAMP-PDE in the FRTL-5 cells through a CAMP-dependent phosphorylation.

A common feature of the hormone-dependent cAMP regulation is that after an initial increase, the cyclic nucleotide concentration returns toward basal levels in spite of the continuous presence of hormone. This phenomenon involves the expression of active intracellular mechanisms terminating the hormonal stimulation, since extracellular dissociation of the hormone from the receptor might not be the primary cause of cessation of a stimulus (1,2). It is also thought to be the result of mechanisms that protect the cell from excessive stimulation (desensitization) or adjust the cell sensitivity to circulating hormone levels (cell adaptation). Uncoupling of the hormone receptor complex from the G,a component of adenylate cyclase, receptor sequestration, and down-regulation are some of the mechanisms causing the termination of the hormonal stimulation andlor desensitization (2-5). Multiple phosphorylations of the receptor by CAMP-independent and CAMP-dependent protein kinases are a primary signal of the uncoupling of the receptor from the transduction system (1, 6, 7).

A decrease in CAMP synthesis might not be the only mechanism causing a return of CAMP to basal levels in the target cell even in the continuous presence of hormone. Using CAMP ana- !ogs that can be distinguished from the endogenous CAMP, i t has been demonstrated that activation of CAMP-dependent protein kinase in adipocytes, hepatocytes, and cardiomyocytes causes a decrease in the endogenous CAMP levels (8, 9). This apparently paradoxical effect is caused by a CAMP-dependent activation of a phosphodiesterase (PDE),’ the enzyme responsible for cAMP degradation (9). This seminal observation opened the possibility that regulation of CAMP hydrolysis plays an important role in terminating the hormonal stimulus and in inducing desensitization in the target cell. A high affinity PDE form, which is rapidly activated by P-adrenergic agonists via a phosphorylation, has been described in platelets and adipocytes (10-13). This form has the properties of a cGMP-inhibited PDE, an enzyme also activated by insulin via a phosphorylation (12, 14, 15). Whether this is the only form regulated in a rapid fashion has been controversial. Reports in the literature

The abbreviations used are: PDE, cyclic nucleotide phosphodiesterase; TSH, thyroid-stimulating hormone; CAMP-PDE, CAMP-specific phosphodiesterase; cGI-PDE, cGMP-inhibited phosphodiesterase; Bt,cAMP, N602’-dibutyryl CAMP; Rolipram, 4-[3-(cyclopentyloxy)-4-me- thoxyphenyll-2-pyrrolidone; Milrinone, 1,6-dihydro-2-methyl-6-oxo- (3,4‘-bipyridine)Wcarbonitrile; TBS, Tris-buffered saline; HB, homogenization buffer; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

9245

9246 Short-term Activation of a Rolipram-sensitive CAMP-PDE

have provided evidence that other PDE isoforms may be regulated by hormones or other stimuli (16-19). In addition to a long term activation of a CAMP-PDE that requires hours to develop and is dependent on altered PDE gene expression (20, 21), there have been several reports suggesting that a CAMP- specific PDE might also be rapidly activated by hormones (18, 22,23). However, lack of biochemical tools to isolate these forms and uncertainties about their properties have left this question largely unanswered. We have recently developed antibodies that specifically recognize the CAMP-PDEs (type IV) (21). In the present study, we have tested the hypothesis that the Rolip- ram-sensitive CAMP-PDE is rapidly regulated by TSH via a phosphorylation.

EXPERIMENTAL PROCEDURES Materials-Coon's modified Ham's F-12 medium (Coon's F-12), bo-

vine insulin and human transferrin, cycloheximide, okadaic acid, and Crotalus atrox snake venom were purchased from Sigma; bovine TSH (1.23 units/mg) for culture was purchased from Armour; Pansorbin cells were purchased from Calbiochem; Immobilon from Millipore; the C subunit of CAMP-dependent protein kinase was purchased from Pro- mega; [2,8-3HlcAMP (20-50 Ci/mmol) and [32Plorthophosphoric acid (8500-9120Ci/mmol) were purchased from DuPont NEN; AG1-X8 resin was purchased from Bio-Rad; ECL Western blot detection kit was purchased from Amersham; Milrinone (1,6-dihydro-2-methyI-6-0~0-(3,4'- bipyridine)-5-~arbonitrile) and Rolipram (4-[3-(cyclopentyloxy)-4-me- thoxyphenyll-2-pyrrolidone) were provided by Syntex. Except where otherwise designated, all other chemicals were the purest grade avail- able from Sigma.

Cell Culture-FRTL-5 cells (ATCC CRL83051, a line of rat thyroid follicular cells developed by Drs. F. S. Ambesi-Impiombato et al. (241, were generously provided by Dr. Leonard Kohn (Section of Cell Regu- lation, NIDDK) and the Interthyr Research Foundation, Baltimore, MD. Cells were routinely cultured in Coon's F-12 medium supple- mented with 5% calf serum and a mixture of three hormones (3H) including TSH (1 milliunit/ml), insulin (10 pg/ml), and transferrin (5 pg/ml). Cells were cultured in 75-cm2 flasks (Corning) at 37 "C in an atmosphere of 95% air, 5% CO, in a humidified incubator.

Stimulation of the PDE Actiuity-Cells were seeded in 35-mm or 90-mm dishes (Corning) in Coon's F-12 containing 5% calf serum and 3H. Five to seven days later, cell monolayers were rinsed twice with Hanks balanced salt solution, and cultures were continued for an additional 24 h with Coon's F-12 medium containing 0.1% bovine serum albumin until the cells became quiescent. The medium was changed 1 h before the experiment. Part of the cells were then incubated for either 15 min in the absence or presence of 10 nM TSH, 1 mM Bt,cAMP, or 30 min in the absence or presence of 100 p~ forskolin. In some experiments, cells were preincubated for 15 min either in the presence of 0.8% Me,SO or 1 p~ okadaic acid dissolved in Me2S0, followed by TSH treatment. TSH stimulation of the PDE activity was also measured after a 4-h preincubation with cycloheximide (15 pg/ml). Cells were harvested in a homogenization buffer (HB) containing 20 mM Tris-C1, pH 8.0, 1 mM EDTA, 0.2 mM EGTA, 50 mM NaF, 10 mM sodium pyro- phosphate, 50 mM benzamidine, 0.5 pg/ml leupeptin, 0.7 pgiml pepsta- tin, 4 pg/ml aprotinin, 10 pgiml soybean trypsin inhibitor, and 2 mM phenylmethylsulfonyl fluoride. Cells were homogenized and centrifuged for 10 min a t 14,000 x g. Both the homogenates and the soluble extracts were used for PDE assay. In some experiments, soluble extracts were subjected to immunoprecipitation.

PDE Assay-PDE activity was measured using 1 PM CAMP as substrate, according to the method of Thompson and Appleman (25) and as detailed previously (17). Samples were assayed in a total volume of 200 pl of reaction mixture including 40 mM Tris-CI, pH 8.0, 10 mM MgCI,, 1.25 mM 2-mercaptoethanol, 1 p~ CAMP, 0.14 mg of bovine serum albumin, and 1 p~ I3H1cAMP (0.1 pCi). In some experiments, 10 p~ Rolip- ram or 10 p~ Milrinone (final concentration) was added to the reaction mixture. After incubation at 34 "C for 5-15 min, the reaction was terminated by adding an equal volume of 40 mM Tris-C1, pH 7.5, containing 10 mM EDTA, followed by heat denaturation for exactly 1 min at 100 "C. To each reaction tube, 50 pg of C. atroz snake venom was added, and the incubation was continued at 34 "C for 20 min. The reaction products were separated by anion exchange chromatography on AGl-X8 resin, and the amount of radiolabeled adenosine collected was quantitated by scintillation counting. Protein concentrations of the samples were measured according to Bradford (26).

Immunoprecipitation-The soluble extracts from control or TSH- treated FRTL-5 cells were precipitated in different incubation conditions using fixed Staphylococcus aureus cells (Pansorbin). Samples were incubated with the Pansorbin alone or plus an anti-CAMP-PDE antiserum (K116), the same antiserum preincubated with the immunogenic peptide (P2224), and a preimmune serum, by procedures described previously (21). PDE activity was measured both in the resuspended pellets and the supernatants of the immunoprecipitation. Adsorbed proteins were then eluted with 1% SDS in phosphate-buffered saline and prepared in a final concentration of 1 x sample buffer for Western blot or autoradiography analyses.

Western Blot Analysis-Immunoprecipitated samples were prepared in 1 x sample buffer (62.5 mM Tris-Cl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 0.7 M 2-mercaptoethanol, and 0.0025% (w/v) bromphenol blue). The samples were boiled for 5 min and subjected to electrophoresis on an 8% SDS-polyacrylamide gel (SDS-PAGE). The proteins were then blotted onto Immobilon membrane followed by blocking the membrane in a TBS solution (20 mM Tris, 14 mM NaCI, pH 7.6) containing 5% bovine serum albumin and 0.1% Tween 20. After several washes, the membrane was incubated with the primary antibody, K116, K116 + P2224, or M3S1, in a TBS solution containing 0.1% Tween 20 (TBS-T). Antiserum K116 was raised against a synthetic peptide of rat PDE3 located at the amino acid positions 105 to 126 (P2224). The antiserum has been characterized previously (21). M3S1 is a monoclonal antibody raised against a fusion protein obtained linking the glutathione trans- ferase to the carboxyl-terminal region of rat PDE3., After a 90-min incubation, the membrane was washed in TBS-T followed by a 1-h incubation with peroxidase-conjugated goat anti-rabbit or goat anti- mouse antibodies a t a dilution of 1:5000 in TBS-T. After washing with the same buffer, the membrane was incubated for 1 min with the ECL detection reagents (Amersham) and exposed to a XAR-5 x-ray film (Kodak) to detect the peroxidase-conjugated secondary antibody for 5-60 s.

Metabolic Labeling with P2P10rthophosphate-FRTL-5 cells were seeded in 90-mm dishes (Corning) and cultured as described above. After induction of quiescence, the medium was replaced with phosphate-free minimal essential medium containing 20 m~ Hepes, pH 7.4, and carrier-free [""Plorthophosphate (0.2-0.3 mCi/ml), and cells were incubated for an additional 2 h. During the last 15 min of incubation, part of the cells were treated with 10 nM TSH. At the end of the treatment, cells were washed three times with Hanks balanced salt solution, harvested in HB, and homogenized. Supernatants from a 10-min centrifugation a t 14,000 x g were incubated batchwise for 30 min with DEAE-cellulose buffered with 200 mM sodium acetate, pH 6.5. The resin was washed twice with the same buffer, and the adsorbed proteins were eluted with 600 mM sodium acetate, pH 6.5. Parallel experiments were carried out to determine the necessary ionic strength to elute the CAMP- PDE activity. These partially purified samples were immunoprecipitated with K116 adsorbed to Pansorbin preincubated with unlabeled soluble extract from quiescent FRTL-5 cells for 1 h, in order to reduce the nonspecific binding of labeled proteins (10). After five to eight washes, the immunoprecipitated proteins were eluted in 1% SDS in PBS, then diluted in SDS-PAGE sample buffer, and separated on a 8% SDS-PAGE. The proteins were blotted onto an Immobilon membrane, and the membrane was dried and exposed to XAR-5 x-ray film.

Protein Kinase Assay-200 pl of the homogenates or the soluble extracts from quiescent FRTL-5 cells were incubated for 5 min in the absence or presence of the catalytic subunit of CAMP-dependent protein kinase (0.2 p ~ ) (Promega). The assay buffer contained 40 mM Tris-CI, pH 7.4, 20 mM magnesium acetate, 0.2 mM ATP. At the end of the incubation, samples were either assayed for the PDE activity or immunoprecipitated with K116 and then assayed for the PDE activity in the absence or presence of 10 PM Rolipram. In some experiments, the immunoprecipitated proteins were separated on a SDS-PAGE and analyzed in Western blot. In the time course study, 100 pl of the soluble extracts from quiescent FRTL-5 cells were incubated in the presence or absence of 0.2 mM ATP and in the presence or absence of a protein kinase inhibitor (2 pg/ml) (27). The assay buffer was the same as described for the PDE assay, containing also 20 mM magnesium acetate, and the final volume in each tube was 2 ml. 200-1.11 aliquots of the reaction mixtures were withdrawn at different times, and the reaction was terminated as described for the PDE assay. After 5 min of incubation, the catalytic subunit of CAMP-dependent protein kinase (0.01 p ~ ) was added to some of the tubes, and the reaction and the PDE assay were carried out as described above.

* S. Iona, M. Cuomo, C. Sette, M. T. Hess, and M. Conti, manuscript in preparation.

Short-term Activation of a Rolipram-sensitive CAMP-PDE 9247

TABLE I Short-term stimulation of the PDE activity in FRTL-5 cells

presence of either 10 nM TSH or 1 m~ BtzcAMP or 30 min in the Quiescent FRTL-5 cells were incubated for 15 min in the absence or

presence or absence of 100 p~ forskolin. At the end of the treatment, cells were washed and homogenized, and PDE activity was determined in the homogenates. n = number of experimental observations, p versus control.

Treatment PDE activity n P

pmoliminimgprotein

Control 75.88 2 2.64 22 TSH 105.72 2 2.06 13 <0.0001 Bt,cAMP 149.86 2 2.69 6 <0.0001 Forskolin 131.57 t 3.26 6 <0.0001

180 I 1

.-

150

30 *L 20 40 60 80 100 24h

TIME (min) FIG. 1. Time course of the PDE activation by TSH. Quiescent

FRTL-5 were incubated for different times in the absence (open circles) or presence (solid circles) of 10 nM TSH. At the end of the incubation, cells were rinsed twice, harvested, and homogenized as described under "Experimental Procedures." The PDE activity was determined directly in the homogenates using 1 PM CAMP as substrate. Each point repre- sents the mean ? S.E. of at least three different experiments, each carried out in triplicate.

RESULTS

Effect of TSH on the PDE Activity in FRTL-5 Cells-TSH induces a rapid increase of the intracellular CAMP levels in FRTL-5 cells. This effect is transient, and, within 15 min, CAMP returns to basal level^.^ In order to understand the mechanism responsible for this transient accumulation of CAMP, the effect of TSH on the PDE activity in the FRTL-5 cells was investigated. Quiescent FRTL-5 cells were treated for 15 min with 10 nM TSH, and the PDE activity was measured in the cell homogenate. In the more than 40 experiments performed, TSH always produced a rapid increase in the PDE activity of these cells (Table I). Treatments with 1 mM Bt,cAMP, a CAMP analog, o r 100 p~ forskolin, an activator of adenylate cyclase, produced an even larger stimulation of the PDE activity, indicating that the effect of the hormone is probably mediated by CAMP (Table I). A 4-h preincubation with 15 pg/ml cycloheximide to prevent de novo synthesis of proteins did not affect the hormonal stimulation of the PDE activity (control = 66.31 5

0.67 pmoVmin x mg, control + cycloheximide = 65.83 2 1.80 pmoVmin x mg, TSH = 112.99 5 1.20 pmoVmin x mg, TSH + cycloheximide = 104.43 f 5.73 pmoVmin x mg; mean 5 S.E. of three different experiments), suggesting that the activation is mediated by a post-translational modification of the enzyme(s). To study the time course of this stimulation, quiescent FRTL-5 cells were treated with 10 nM TSH for different times up to 24

S. Takahashi, J. V. Swinnen, J. J. Van Wick, and M. Conti, manuscript in preparation.

'*O '

TSH concentration (nM)

the PDE activity of FRTL-5 cells. Quiescent FRTL-5 were incubated FIG. 2. TSH concentration dependence of the stimulation of

for 15 min with increasing concentrations of TSH. At the end of the incubation, cells were harvested, and the PDE activity was determined as described in Fig. 1. The ED,, calculated from the curve is 4 x lo-" M. The data are the mean t S.E. of three different experiments.

TABLE I1 Effect of type III and type N specific PDE inhibitors on

TSH-stimulated PDE activity Quiescent FRTL-5 cells were incubated for 15 min in the absence or

presence of 10 nM TSH, cells were harvested in HB, and homogenized, and the PDE activity of the cell extracts was determined. The PDE assay was performed in the absence or presence of either 10 PM Rolip- ram or 10 PM Milrinone (final concentration) in the reaction mixture.

Treatment PDE activity

No inhibitor Rolipram (IO p ~ ) Milrinone (10 p ~ )

pmol imin img protein Control 56.93 2 1.09 20.69 2 1.64 TSH

37.56 t 4.20 88.33 2 6.18 21.77 2 1.64 55.45 -c 0.18

h, and the homogenate PDE activities were measured. This time course study showed that the hormonal activation was already detected after 3 min, reached a maximum in 15 min, then remained elevated for 90 min. A delayed increase after 24 h was also observed (Fig. 1). This delayed stimulation has been previously characterized and is dependent on de novo PDE synthesis (28L3 The dose dependence curve indicated that TSH exerted its maximal effect at a concentration of 1 nM, EDs0 = 4 x lo-'' M (Fig. 2). These hormone concentrations are compa- rable to those required to stimulate DNA synthesis and phosphorylation of several substrates (29, 30).

Characterization of the PDE Activity Stimulated by TSH- The PDE inhibitors Milrinone, specific for the class I11 PDE (cGMP-inhibited PDE) (31, 32), and Rolipram, specific for the class IV PDE (CAMP-specific PDE) (31, 33, 341, were used to determine the properties of the PDE activated by TSH (Table 11). Both the basal and stimulated PDE activities of the soluble fraction were inhibited by Rolipram, while Milrinone was less effective and did not affect the hormonal stimulation. To further characterize the PDE form that is stimulated by TSH, cell extracts from control and TSH-treated cells were incubated with an anti-CAMP-PDE (K116) preadsorbed to Pansorbin., This antibody immunoprecipitates type IV PDEs, but not the 61- and 63-kDa calmodulin-dependent PDEs or the CGI-PDE.~.~ The PDE activity of both the adsorbed and the supernatant fractions was determined in the presence or absence of 10 p~

M. Conti, unpublished results


I r

I st 3Lm Z% m a

Rolipram in the PDEassay -

d - + - + “ -

1

lK116 K116 K116 K116 NO K116 AB + ~ 2 2 2 4 serum I

IMMUNOPRECIPITATION

FIG. 3. Immunoprecipitation of the TSH-stimulated PDE activity. Quiescent FRTL-5 were incubated for 15 min in the absence or presence of 10 nM TSH. At the end of the incubation, cells were rinsed and harvested as described under “Experimental Procedures.” Soluble extracts were incubated for 90 min with Pansorbin alone, Pansorbin preadsorbed to K116, to K116 plus peptide 2224, or to a preimmune serum. At the end of the incubation at 4 “C, samples were centrifuged to separate free and immunoadsorbed PDE activity. The activity recovered in the supernatant and in the pellet of the immunoprecipitation was measured using 1 PM CAMP as substrate in the absence or presence of 10 PM Rolipram. Each bur is the mean of a t least three different experiments.

Rolipram. Immunoprecipitation of the same samples was also performed using either Pansorbin alone or the anti-CAMP-PDE antiserum preadsorbed to the immunogenic peptide (P2224), or a preimmune serum, to determine the specificity of the antibody-PDE interaction. The results reported in Fig. 3 indicate that the TSH-stimulated PDE activity is quantitatively immunoprecipitated by the CAMP-PDE specific antibody (K116), while the activity remaining in the supernatant was not affected by TSH. Approximately 90% of the activity recovered in the immunoprecipitation pellet was inhibited by 10 p~ Rolip- ram. Neither Pansorbin alone nor the antibody preadsorbed to the synthetic peptide nor the preimmune serum precipitated significant PDE activity (Fig. 31, indicating that the activity recovered in the immunoprecipitation pellet is entirely due to specific interactions of the PDE with the CAMP-PDE antibody.

Effect of TSH on the Physicochemical Properties of a CAMP- PDE-The possibility that a phosphorylation is involved in the TSH stimulation of a PDE was tested using okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A (35, 36), together with TSH. It has been reported that okadaic acid treatment causes activation of a cGI-PDE similar to that observed with insulin in rat adipocytes (37). When quiescent FRTL-5 cells were preincubated for 15 min with 1 p~ okadaic acid and then treated with 10 nM TSH for 15 min, the stimulation was higher than that in cells treated with TSH alone ( p < 0.0001), while okadaic acid by itself did not significantly affect (p > 0.005) the PDE activity (Table 111). This result

TABLE I11 Okadaic acid potentiation of the TSH effect on the PDE activity of

FRTL-5 cells Quiescent cells were preincubated for 15 min either with 0.8% Me2S0

or 1 J ~ M okadaic acid dissolved in Me2S0. Part of the cells were then treated with 10 nM TSH for an additional 15 min. At the end of the incubation, cells were harvested and homogenized, and the PDE activ-

observations; p uersus control. ity was determined in the homogenates. n = number of experimental

Treatment PDE activity n P

pmol lminlmg protein Control TSH

66.54 ? 1.21 8 91.83 t 2.10” 10 <0.0001

Okadaic acid 73.60 ? 1.89 Okadaic + TSH

8 >0.005 108.12 t 3.44” 8 <0.0001

“ The difference between these two values is significant ( p < 0.001).

suggested that inhibition of dephosphorylation has a synergis- tic effect on the TSH stimulation of the PDE activity in these cells. To determine whether the TSH activation was associated with a change in the pattern of PDE expression in FRTL-5 cells, Western analysis was performed on extracts from control and TSH-treated cells. Soluble extracts from control cells and TSH- treated cells were immunoprecipitated under different conditions as described in Fig. 3. The adsorbed proteins were fractionated by SDS-PAGE, and Western blot analyses were performed using two different CAMP-PDE antibodies, K116 and M3S1. When the soluble extracts were immunoprecipitated with K116, the Western blot analysis with the antibody re- vealed a predominant polypeptide with apparent M , of 92.6 2

0.8. Three less abundant polypeptides with apparent M , of 109.3 A 1.9, 72.0 A 1.2, and 67.3 2 0.5 (mean 2 S.E. calculated on three different SDS-PAGES) were detected in control cells (Figs. 4A and 5). A 15-min TSH treatment had no effect on the 67-, 72-, and 109-kDa polypeptides, but caused the appearance of an additional band with M , of 99.4 2 1.60 (mean 2 S.E. calculated on three different SDS-PAGES). The appearance of this band was associated with a decrease in the intensity of the 93-kDa band (Fig. 4A), suggesting that the 99-kDa band is the result of an altered migration of the 93-kDa polypeptide. The abundance of the 93-kDa immunoreactive polypeptide closely followed the PDE activity, since it was not present when the immunoprecipitating antibody was omitted, when replaced with a preimmune serum, or preadsorbed with peptide 2224 (Fig. a), conditions that did not cause the precipitation of appreciable PDE activity (Fig. 3). Under these conditions, only the immunoprecipitation of the 109-kDa band was incom- pletely blocked by preadsorption (Fig. 4A). To further investi- gate the nature of the immunoreactive polypeptides and the specificity of the interaction with the antibody, Western blot analyses were performed on soluble extracts from control cells, cells treated for 15 min or 24 h with TSH, and on a partially purified CAMP-PDE from unstimulated cells (Fig. 4, B and C). These data demonstrate that the 93-99-kDa doublet was present after both 15-min and 24-h treatment with TSH, while the intensity of the 72- and 67-kDa bands were markedly increased after long term treatment with the hormone. This increase is in agreement with our previous observation that this set of CAMP- PDEs is induced after long term treatment with hormones (21). Furthermore, a partial purification of the cytosolic proteins from FRTL-5 cells using an ion exchange HPLC chromatography, showed that a major immunoreactive polypeptide of the same size as the polypeptide affected by the short-term TSH treatment co-purified with the PDE activity (Fig. 4, B and C). All these bands, with the possible exception of the 109-kDa band, were absent when the antiserum was preadsorbed during the immunoprecipitation or the Western blot (Fig. 4, B and C). The 93- and 99-kDa polypeptides were also recognized by a


A IMMUNOPRECIPITATION I 2 -

0 +

200 - 1 - kDa

200 - 1

WESTERN

K116

97.4 - : 110 7 100 ' 93 c_ 67

97.4 - " - " 68 -

46 - ..010 FIG. 5. Western blot analysis of the CAMP-PDE in FRTL-5 in the

presence of cycloheximide. Cells were preincubated for 4 h in the absence or presence of 15 pg/ml cycloheximide (CHX) . Following the preincubation, cells were incubated for 15 min in the absence or presence of 10 nM TSH. Immunoprecipitation and preparation of the samples for the SDS-PAGE were performed as described in Fig. 3. Immunoreactive bands were analyzed in Western blot using the CAMP- PDE antibody K116.

IMMUNOPRECIPITATION

' K116 +P2224 ' K116

monoclonal antibody raised against a carboxyl-terminal epitope of a CAMP-PDE (M3S1), further supporting the hypothesis that these polypeptides are indeed CAMP-PDEs (Fig. 40). The TSH induction of the 99-kDa band was not due to a de nouo protein synthesis, since preincubation of the cells with cycloheximide (15 pg/ml) did not prevent the band shift (Fig. 5).

It has been shown that phosphorylation of serine, threonine, and tyrosine amino acidic residues can alter the electrophoretic behavior of a protein, resulting in decreased mobility (38-42). To determine whether the appearance of the new band was associated with a phosphorylation of the PDE polypeptide, in vivo labeling of the PDE was performed. Quiescent FRTG5 cells were labeled for 2 h with [32PJorthophosphate, and half of the cells received TSH in the last 15 min of incubation. Soluble extracts from control and TSH-treated cells were partially purified by anion exchange chromatography (DEAE-cellulose), and immunoprecipitation with K116 was performed as described under "Experimental Procedures." The incorporation of 32P into the protein was determined by SDS-PAGE followed by autoradiography. Although the resolution of this autoradiography did not allow the separation of the 93-99-kDa doublet, in the three different experiments performed, TSH treatment led to an increased incorporation of 'jZP in a band of apparent M, =

WESTERN

K116

B

C

- kDa

200 - 97.4 -

68 - 46 -

- kDa

200 - 97.4 -

68 - 46 -

- -93 -1 00

K116

+ P2224 v

-93

- IgG

D IMMUNOPRECIPITATION

were harvested as described in Fig. 1, and homogenates were centrifuged for 10 min a t 14,000 x g. Soluble extracts were immunoprecipitated under different conditions for 90 min (see Fig. 3 for details). The immunoadsorbed proteins were separated by centrifugation and eluted with 1% SDS in phosphate-buffered saline, diluted in sample buffer, and separated by SDS-PAGE. Western blot analysis was performed with the CAMP-PDE antibody K116. B and C, quiescent cells were incubated for 15 min or 24 h in the presence or absence of 10 ny TSH. Soluhlc extracts were immunoprecipitated with either K116 or K116 preadsorbed to the peptide 2224 used as immunogen. The adsorbed proteins were eluted as described above and fractionated by SDS-PAGE. Western blot analyses were performed either with K116 (B) or K116 preadsorbed to peptide 2224 (C). A partially purified CAMP-PDE from quiescent FRTL-5 was loaded in the last lanes of both gels (HPLC). D, quiescent cells were treated a s described in A. Solublc extracts were precipitated with either Pansorbin alone or Pansorbin + K116. The adsorbed proteins were fractionated by SDS-PAGE. The Western blot analysis was performed with the monoclonal CAMP-PDE antibody M3S1. The molecular size markers used in all gels were myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), and ovalbumin (46 kDa).

kDa kDa - - 200 - ' /-

WESTERN

97.4 - ' - 5 3 100

M3S1

68 - I

FIG. 4. Western blot analysis of the CAMP-PDEs present in FRTL-5 cells. A, quiescent cells were incubated for 15 min in the absence or presence of 10 ny TSH. At the end of the incubation, cells


200 -

97.4 -

68 -

46 -

PDE

FIG. 6. Effect of TSH on protein phosphorylation in FRTC5 cells. Quiescent FRTL-5 were preincubated with I.‘v2Plorthophosphate for 90 min before treatment for 15 min in the absence or presence of 10 nhl TSH. At the end ofthe incubation, cells were washed three times and harvested as described in Fig. 1, and the soluble extracts adsorbed to DEAF,-cellulose. After this partial purification by ion-exchange chromatography (see “Experimental Procedures” for a detailed description), the eluted proteins were incubated for 90 min with Pansorbin alone, Pan- sorbin preadsorbed to K116, or to K116 plus peptide 2224. The immunoadsorbed proteins were separated by centrifugation and by SDS- PAGE and transferred to an Immobilon membrane. The membrane was dried and analyzed by autoradiography. The arrows on the right of the picture indicate the position of the doublet recognized by K116 in the Western blot analysis of the same membrane.

90-99 (Fig. 6). This band co-purified with the enhanced PDE activity both on DEAE-ion exchange chromatography and immunoprecipitation. The TSH-stimulated band was absent when the antibody was omitted or was preadsorbed with the peptide used for immunization (data not shown). Experiments in which the DEAE-step was omitted and the immunoprecipitation was repeated twice gave identical results (data not shown).

In Vitro Stimulation of the PDE Activity by CAMP-dependent Protein Kinase-To determine whether CAMP-dependent protein kinase mediates the TSH-dependent stimulation of CAMP- PDE, the activation of the PDE in a cell-free system was investigated. Soluble extracts from quiescent FRTL-5 cells were incubated in the presence or absence of the catalytic subunit of CAMP-dependent protein kinase, and the PDE activity was determined at different times after the addition of the kinase. As shown in Fig. 7A, CAMP-dependent protein kinase produced a 2-3-fold stimulation of the rate of cAMP hydrolysis. The CAMP-dependent protein kinase stimulation was abolished by the addition of a synthetic CAMP-dependent protein kinase inhibitor (Fig. 7 A ) . Incubation of the cell extract with the kinase in the absence of ATP produced only a minor increase in cAMP hydrolysis, confirming that the kinase effect requires the presence of ATP (Fig. 7A ). Furthermore, immunoprecipitation of the reaction mixture with K116 indicated that all the CAMP- dependent protein kinase-stimulated PDE activity was immunoprecipitated by the antibody and that 90% of the immunoprecipitated activity was inhibited by 10 p~ Rolipram, while the activity not immunoprecipitated was only slightly affected by the inhibitor (Fig. 7B). Furthermore, SDS-PAGE and West- ern blot analysis of the cytosol incubated in the absence or presence of CAMP-dependent protein kinase demonstrated the appearance of the doublet as observed in the intact cells treated with TSH (Fig. 8). From this experiment we concluded that CAMP-dependent protein kinase is able to activate in vitro a CAMP-PDE with immunological properties similar to the CAMP-PDE stimulated in the intact cell by TSH. Similar results were obtained after partial purification of the CAMP-PDE

IMMUNOPRECIPITATION

FIG. 7. Stimulation of the CAMP-PDE activity by c. AMP-dependent protein k inase in a cell-free system. Quiescent FRTL-5 cells were harvested as described in Fig. 1. A, soluble extracts (100 pl) were diluted with the PDE assay reaction buffer, containing 20 mM magnesium acetate, in the absence or in the presence of 0.2 mal ATP, and in the absence or in the presence of a CAMP-dependent protein kinase inhibitor (see “Experimental Procedures”). After 5 min of incubation, the catalytic subunit of CAMP-dependent protein kinase (0.01 p ~ ) was added to part of the tubes. Open circles represent soluble extract +ATP solid circles represent soluble extract + CAMP-dependent protein kinase; solid .squares represent soluble extract + ATP + CAMP- dependent protein kinase: solid triangles represent soluble extract + ATP + CAMP-dependent protein kinase + CAMP-dependent protein kinase inhibitor. B, soluble extracts from quiescent FRTL-5 cells (200 pl) were incubated for 5 min in the absence or presence of the catalytic subunit of CAMP-dependent protein kinase (0.2 PM). At the end of the incubation, the PDE activity was measured, the rest of the samples were immunoprecipitated with K116, and the PDE activities were measured both in the resuspended pellets and the supernatants, in the absence or presence of Rolipram as detailed in Fig. 3.

by HPLC-DEAE-ion exchange chromatography (data not shown).

DISCUSSION

The experiments described demonstrate that a CAMP-specific, Rolipram-sensitive PDE expressed in FRTL-5 thyroid cells is activated within minutes of exposure of the cells to TSH. This regulation is mediated by a CAMP-dependent activation of CAMP-dependent protein kinase and is associated with the


200 - 1 ’ 110 “1 97.4 - - - =<- 100

93 68 - , - -~ 67

46- - FIG. 8. Western blot analysis of the cAMP-dependent protein

kinase-stimulated CAMP-PDE of FRTL-5 cells. Soluble extracts from quiescent FRTL-5 were incubated for 5 min in the absence or in the presence of the catalytic subunit of CAMP-dependent protein kinase. At the end of the incubation, samples were immunoprecipitated with K116 as described under “Experimental Procedures.” The adsorbed proteins were separated on a SDS-PAGE, and Western blot analysis with the same antibody was carried out as described in Fig. 4.

phosphorylation and altered mobility of a polypeptide with the same properties of a CAMP-PDE. These data, together with the cell-free activation of a PDE with CAMP-dependent protein kinase, strongly suggest a causative link between phosphorylation and activation of a CAMP-PDE. This short-term activation of a PDE and the consequent increase in CAMP degradation contributes to the termination of the hormone stimulation and, together with the long-term regulation of the CAMP-PDE expression, is part of the mechanisms that mediate cell adaptation to external stimuli.

A short-term activation of a PDE has been previously observed in several systems. A cGI-PDE is stimulated by insulin and isoproterenol in rat adipocytes (12, 14) and by insulin (15) and prostaglandins (10, 11) in human platelets. Direct (10, 12, 14, 15) and indirect (11, 13, 37) evidence indicate that hormones activate a cGI-PDE through a phosphorylation. The effect is at least in part mediated by the CAMP-dependent protein kinase, since it can be reproduced in a cell-free system by the addition ofATP and the catalytic subunit of the kinase (13) and is inhibited in vivo by CAMP-dependent protein kinase inhibitors (11). The following findings support our conclusion that a CAMP-PDE and not a cGI-PDE is rapidly activated by TSH in FRTL-5 thyroid cells. Unlike the cGI-PDE activated in adipocytes which is bound to the microsomal cell fraction, the PDE activated by TSH was exclusively recovered in the soluble fraction of the cells. Furthermore, the TSH-regulated PDE was sensitive to Rolipram but not Milrinone, a specific inhibitor for the cGI-PDE (type I11 PDEs) (31, 32). Finally, the hormone- sensitive PDE present in FRTL-5 cells was quantitatively immunoprecipitated by a n antibody specific for the Rolipram- sensitive PDE. This antibody was raised against a peptide sequence absent in any of the cGI-PDE sequences thus far reported. At the concentration used, this antibody did not immunoprecipitate a partially purified cGI-PDE derived from HL60 c e k 4 The whole of these data therefore indicate that TSH activates a Rolipram-sensitive CAMP-PDE.

Although we have reported that pituitary hormones regulate the PDE activity of Sertoli cells and FRTL-5 cells via regulation of the CAMP-PDE mRNAs (20, 21, 28), the rapid increase in PDE activity described here is not due to de novo synthesis of a CAMP-PDE, since preincubation of the cells with cycloheximide did not affect significantly the extent of the stimulation. Furthermore, an activation is observed as early as 3 min after hormone addition, and, at this time, no increase in CAMP-PDE

mRNA could be detected. These experiments indicate that a post-translational modification is the cause, directly or indi- rectly, of the PDE activation. The TSH-dependent activation of the CAMP-PDE activity was associated with the altered mobility of a polypeptide of 9 3 kDa. This polypeptide is recognized by polyclonal and monoclonal antibodies raised against two different epitopes of a CAMP-PDE. The molecular weight of this polypeptide is also similar to a CAMP-PDE expressed in eu- karyotic cells. On the basis of these findings, we propose that the 93-99-kDa polypeptide corresponds to a CAMP-PDE. The decreased mobility of this band after TSH treatment suggests that the PDE itself is modified by the TSH treatment. Since proteins were separated under reducing and denaturing conditions, it is unlikely that the shift of the band is due to an interaction of the CAMP-PDE with other polypeptides through noncovalent bonds. Therefore, the most plausible explanation of our data is tha t a post-translational covalent modification is the cause of the altered mobility of the CAMP-PDE polypeptide.

There are numerous reports in the literature indicating that the phosphorylation of a polypeptide alters the migration on SDS-PAGE (38-42). The following data support the idea that the altered mobility of the CAMP-PDE polypeptide is caused by a TSH-dependent, CAMP-dependent protein kinase-mediated phosphorylation. The increase in szP incorporation in the polypeptide tha t co-purifies with the CAMP-PDE in the immu- noaffinity adsorption and after a DEAE-ion exchange chromatography is consistent with the hypothesis of a rapid phosphorylation of a CAMP-PDE. The fact that the activation of a CAMP-PDE and the appearance of the 93-99-kDa doublet can be reproduced by incubating soluble extracts of FRTL-5 cells with the catalytic subunit of the CAMP-dependent protein kinase is also in agreement with this hypothesis. Our data, however, do not provide direct evidence of a causative link between the phosphorylation-altered migration of the CAMP-PDE and the increase in catalytic activity. Phosphorylation experiments in a cell-free system with purified CAMP-PDE should provide a definitive answer to this question. Finally, whether the catalytic subunit of CAMP-dependent protein kinase phosphory- lates directly a CAMP-PDE remains to be determined because intermediate steps and activation of other kinases might be involved in the PDE-altered motility and phosphorylation.

Previous reports have shown that in skeletal myoblasts and adult muscle extracts Bt,cAMP induced both a rapid activation of pre-existing PDEs and their de novo synthesis (18, 19). The data presented herein provide immunological evidence of a rapid activation of the CAMP-PDEs by hormones that increase CAMP. This together with our previous observation (28) indi- cates that both short-term and long-term regulations of CAMP- PDEs operate in the FRTL-5 thyroid cells. Madelian and La Vigne (23) have reported that in astroglia cells isoproterenol produces the rapid activation of a PDE with properties similar to a CAMP-PDE. A rapid activation of a CAMP-PDE has also been observed in rat thymocytes after concanavalin A treatment probably via a CAMP-independent mechanism (22). Al- though the nature of these forms and the mechanism of the activation were not defined, it is probable that regulations similar to that observed in FRTL-5 cells is occurring in other cells.

Four different genes coding for CAMP-PDE have now been identified in the rat (20, 43), and their expression is cell- and tissue-specific (43). Hormonal regulation of the mRNA level and of the protein expression of some of these isoforms has been reported (20, 21, 28). At present we have no conclusive infor- mation on the form that is activated in FRTL-5 cells within minutes of TSH stimulation. Since both antibodies used have a higher affinity for the rat PDE3flVd protein, it is possible that this is the form activated by TSH. Experiments with recombi-

9252 Short-term Activation of a Ro

nant enzymes expressed in heterologous systems should clarify this point.

In summary, our results indicate that a CAMP-PDE can be rapidly activated by hormones and CAMP through a CAMP- dependent protein kinase-dependent phosphorylation. In those cells where a CAMP-PDE is expressed at the same time as a cGI-PDE, both enzymes are activated upon the increase in CAMP levels and CAMP-dependent protein kinase activation. Both enzymes, therefore, should contribute to the short-term feedback loop that regulates CAMP levels in the hormone-target cell.

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