©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Site in the cGMP-inhibited Phosphodiesterase Phosphorylated in Adipocytes in Response to Insulin and Isoproterenol (*)

(Received for publication, December 5, 1995; and in revised form, January 19, 1996)

Tova Rahn Lars Rönnstrand (3) Marie-Josephe Leroy (2) Christer Wernstedt (3) Hans Tornqvist (1) Vincent C. Manganiello (2) Per Belfrage Eva Degerman (§)

From the  (1)Department of Cell and Molecular Biology, Section for Molecular Signalling, Lund University, Sweden and theDepartment of Pediatrics, University Hospital, S-221 00 Lund, Sweden, (2)NHBLI, National Institutes of Health, Bethesda, Maryland 20892, and the (3)Ludwig Institute for Cancer Research, Biomedical Center, S-751 85 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C. J., Tornqvist, H., Vasta, V., Manganiello, V. C., and Belfrage, P.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of MFRRPS(P)LPCISREQ. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic P-peptides showed that a single site in cGI PDE (Ser) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C.(1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).


INTRODUCTION

Insulin controls cell metabolism and proliferation through several multicomponent diverging pathways, which are only partially understood (1, 2, 3) . One important and well known metabolic effect is to antagonize hormone-activated lipolysis in adipose tissue, brought about mainly via insulin-mediated activation of a membrane-associated cGMP-inhibited phosphodiesterase (cGI PDE, (^1)referred to as the PDE 3 gene family; (4) ), which leads to a reduction in cAMP, a decrease in cAMP-dependent protein kinase (protein kinase A) activity, and a lowering of the phosphorylation and thereby the activity of the hormone-sensitive lipase, the rate-limiting enzyme in the regulation of lipolysis(5, 6, 7, 8, 9, 10, 11) .

Insulin and cAMP-increasing agents induce serine phosphorylation and activation of cGI PDE in rat adipocytes(12, 13) . An insulin-stimulated cGI PDE serine protein kinase activity in rat adipocytes has previously been partially characterized(14, 15) . In the presence of both isoproterenol and insulin, the phosphorylation/activation of cGI PDE is more than additive(13, 16) , suggesting cross-talk between the two signal transduction pathways.

To further dissect the components of the antilipolytic signaling pathway controlling the cGI PDE, we used two-dimensional tryptic phosphopeptide mapping and other methods to analyze the site(s) phosphorylated in this enzyme in P-labeled rat adipocytes incubated with insulin, isoproterenol, or both. Since the amount of labeled phosphopeptides that could be obtained from experiments with intact cells was far less than that required for amino acid sequencing, the phosphorylation site(s) was identified by comparison of properties of phosphopeptides from cGI PDE phosphorylated in adipocytes, or from recombinant cGI PDE expressed in NIH 3006 human fibroblasts (^2)(rcGI PDE) phosphorylated by protein kinase A, with peptides synthesized on the basis of the deduced sequence of rat adipocyte cGI PDE (17) and phosphorylated by protein kinase A. Peptides chosen for these studies contained consensus sequences for phosphorylation by protein kinase A (18) , since preliminary results indicated that the site(s) phosphorylated during incubation of adipocytes with insulin or isoproterenol was located in the same tryptic phosphopeptide.


EXPERIMENTAL PROCEDURES

Phosphorylation and Trypsinization of Synthetic Peptides Corresponding to Rat Adipocyte cGI PDE Amino Acid Sequences and Phosphorylation of Rat Adipocyte and Recombinant NIH-3006 Fibroblast cGI PDE

The peptides VIRPRRRSSCVSLGESAAGYYGSGKMFR (P), MFRRPSLPCISREQ (P), and LRRSSGASGLLTSEHHSR (P) (Fig. 1) were synthesized (Biomolecular Core Facilities, Lund University) on an Applied Biosystems model 430A using the N-(9-fluorenyl)methoxycarbonyl (Fmoc) program and purified by preparative reversed phase chromatography (Kromasil C8 column). We previously reported that the major site in adipocyte cGI PDE phosphorylated by protein kinase A in vitro is Ser(18) , localized within P (Fig. 1). The identity and purity of the synthetic peptides were assessed by amino acid analysis. The peptides were phosphorylated with protein kinase A (Sigma, P-2645), subjected to chromatography on C18 disposable minicolumns (Chromabond; Macherey-Nagel), and vacuum centrifuged(18) . The phosphopeptides (PP) were suspended in 50 µl of 50 mM ammonium bicarbonate buffer and trypsinized with 1 µg of trypsin (modified sequencing grade; Promega) overnight at 37 °C. The digests were subjected to vacuum centrifugation, oxidized, and re-trypsinized as described for the cGI PDE tryptic phosphopeptides below. All tryptic phosphopeptides (tPP) used in this study are presented in Fig. 1.


Figure 1: Linear structure of cGI PDE. Boxes indicate putative protein kinase A consensus sequences; tryptic cleavage sites are marked by arrows. Tryptic fragments derived from protein kinase A-phosphorylated synthetic peptides P and P are denoted tPP/tPP and tPP, respectively. Tryptic fragments derived from protein kinase A-phosphorylated P are not indicated.



Particulate adipocyte cGI PDE, corresponding to 1 ml packed cell volume, was prepared and solubilized(12, 13) . NIH-3006 cells (NIH-3T3 cells that stably overexpress the human insulin receptor; (19) ) were stably transfected with full-length cGI PDE cDNA and cultured as described.^2 Confluent cells were washed rapidly with ice-cold phosphate-buffered saline, suspended in 50 mM Tris, pH 7.4, containing 5 mM MgCl(2), 1 mM EDTA, 5% glycerol, 1% CE (heterogeneous non-ionic detergent; (18) ), 10 µg/ml antipain and leupeptin, and 1 µg/ml pepstatin A (1 ml/dish) and harvested with a rubber policeman. The cell suspension was sonicated (three times for 20 s each, on ice in a Branson Sonifier 250, output control 1.5, constant duty cycle) and thereafter centrifuged (33,000 times g, 60 min, 4 °C). Solubilized adipocyte cGI PDE (360 pmol/min; PDE activity was determined as described previously) (12, 13, 20) or solubilized rcGI PDE (40 nmol/min), derived from one dish, was incubated with 50 mM HEPES, pH 7.4, containing 10 mM MgCl(2), 1 mM dithioerythritol, 40 µM [-P]ATP, and 100 units/ml protein kinase A, in final volumes of 12 or 2 ml respectively, for 30 min at 30 °C.

Tryptic and Asp-N Protease Phosphopeptide Mapping of PcGI PDE

Adipocytes from Sprague-Dawley rats (35-37 days old) were prepared (21, 22) and incubated with P(i) (0.5-1 mCi/ml)(12, 13) . Solubilized cGI PDE from hormone-stimulated P-labeled adipocytes(12, 13) and protein kinase A-phosphorylated cGI PDE (see above) was incubated overnight with anti-P, a polyclonal antiserum raised against P(12, 18) . The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (23) , followed by electroblotting to nitrocellulose membranes (Amersham Life Science) (30 V, 16 h, in 5% methanol, 96 mM glycine, 12.5 mM Tris). Solubilized rcGI PDE was phosphorylated in vitro by protein kinase A and directly subjected to SDS-PAGE, followed by electroblotting to nitrocellulose membranes. P-Phosphorylated cGI PDE was localized by digital imaging of P (Fujix BAS 2000, Fuji Inc., Tokyo, Japan). That portion of the membranes was excised and the phosphoproteins digested with trypsin essentially as described(24) . Briefly, the membrane pieces were incubated for 30 min at 37 °C in 0.6% acetic acid (HAc) and 0.5% polyvinylpyrrolidone, washed three times with distilled water, and incubated with trypsin (1 µg of trypsin in 200 µl of 50 mM ammonium bicarbonate buffer/piece) for 16 h at 37 °C. More than 95% of the P radioactivity was released from the nitrocellulose membranes. The eluates were vacuum centrifuged, and the dried peptides were thereafter oxidized with formic acid (final volume of 50 µl) for 60 min at 4 °C. The oxidized phosphopeptides were diluted in 450 µl of water, frozen and subjected to vacuum centrifugation, carefully dissolved in 50 µl of 50 mM ammonium bicarbonate buffer, and trypsinized for 16 h. After the second trypsinization, 140 µl of pH 1.9 buffer (formic acid:HAc:H(2)O (44:156:1800; v/v/v)) were added to the tryptic digests, and, after centrifugation (10,000 times g, 5 min), 180 µl were subjected to vacuum centrifugation. Finally, the phosphopeptides were dissolved in 7 µl of pH 1.9 buffer and subjected to two-dimensional phosphopeptide mapping(25) , using the Hunter thin layer electrophoresis apparatus (HTLE-7000, CBS Scientific). First dimension thin layer electrophoresis (TLE) was performed in pH 1.9 buffer for 25 min at 2000 V; second dimension ascending thin layer chromatography (TLC) was performed in isobutyric acid buffer (isobutyric acid:n-butanol:pyridine:HAc:H(2)O (1250:38:96:58:558; v/v/v/v/v)). After localization of P by digital imaging, in some experiments P-phosphopeptides were scraped from the TLC plates, eluted in pH 1.9 buffer, and lyophilized. The eluates were then either re-run in the two-dimensional system to assess co-migration with other peptides, immunoprecipitated with anti-P followed by Tricine-SDS/urea-PAGE(26) , or subjected to additional digestion with endoproteinase Asp-N (sequencing grade, 2 µg reconstituted in 50 µl of H(2)O, giving a final concentration of 10 mM Tris; Boehringer Mannheim). Briefly, the eluted tryptic phosphopeptides were suspended in 50 µl of 50 mM ammonium bicarbonate buffer and further digested with 2 µg of Asp-N protease (0.4 µg added 5 times at 2 h intervals, followed by incubation overnight at 37 °C). Trifluoroacetic acid (700 µl of 0.1%) and 1 µg of carrier-P were added and the digests were applied to C18 disposable minicolumns and subjected to two-dimensional phosphopeptide mapping.

Estimation of the Stoichiometry of Hormone-induced Phosphorylation of cGI PDE

cGI PDE (0.06 pmol/sample as estimated from enzyme activity; (13) ), P-phosphorylated in adipocytes as described above, was immunoprecipitated, subjected to SDS-PAGE, and analyzed by digital imaging and volume integration analysis of P. P radioactivity was determined from a standard curve relating volume integration values linearly to in vitro protein kinase A phosphorylated rcGI PDE (2-600 pmol) (P radioactivity obtained by liquid scintillation counting of pieces cut out from SDS-polyacrylamide gels). The specific activity of intracellular [-P]ATP at steady state was assumed to be half that in the extracellular medium (27) . The total in vitro phosphorylation stoichiometry of cGI PDE was determined as in (18) . Phosphorylation stoichiometry for individual tryptic phosphopeptides was estimated by comparing their volume integration values and relating them to total stoichiometry.


RESULTS

Tryptic Phosphopeptide Mapping of cGI PDE Phosphorylated in Adipocytes in Response to Isoproterenol and/or Insulin

In intact adipocytes, the 135 kDa rat adipocyte cGI PDE was maximally phosphorylated to a similar extent in the presence of isoproterenol (Fig. 2A, lane 2) or insulin (lane 3) (12) with an estimated total phosphorylation stoichiometry of 0.2 ± 0.06 (mean ± S.E., n = 5 for each hormone). More than additive phosphorylation (Fig. 2A, lanes 5-7) and activation (data not shown) occurred during incubation with submaximal concentrations of both hormones at shorter incubation times (13, 16) . [P]cGI PDE was immunoprecipitated, subjected to SDS-PAGE, and transferred to nitrocellulose membranes before tryptic digestion and analysis by two-dimensional phosphopeptide mapping. As shown in Fig. 2(B-D), the P-phosphorylated cGI PDE in each case yielded one major tryptic phosphopeptide (tPP, tPP, or tPP) and one minor tryptic phosphopeptide, which in all cases appeared to migrate to the same positions. Similar results were obtained in 18 separate experiments (7 experiments each for isoproterenol or insulin alone and 4 experiments for stimulation by the combination of the hormones). The amount of the minor phosphopeptide varied considerably between experiments and contributed to 0-30% of the total amount of tryptic phosphopeptides. The small amount of this phosphopeptide precluded further analysis.


Figure 2: Two-dimensional tryptic phosphopeptide maps of cGI PDE from intact rat adipocytes, stimulated with isoproterenol and/or insulin. A, solubilized cGI PDE from P-labeled rat adipocytes (2 ml of 10% by volume of cells/condition) stimulated with isoproterenol (ISO), insulin (INS) or in combination (ISO+INS), as indicated, was immunoprecipitated and subjected to SDS-PAGE, followed by digital imaging of P. B-D, solubilized cGI PDE from P-labeled rat adipocytes (20 ml of 10% by volume of cells with 0, 5-1 mCi/ml) stimulated with isoproterenol and/or insulin was immunoprecipitated, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and digested with trypsin. P-Phosphopeptides were separated by two-dimensional TLE/TLC and visualized by digital imaging of P. The major tryptic phosphopeptides are indicated as tPP, tPP, or tPP. Minor tryptic phosphopeptides are indicated with arrows. X marks the origin. The electrophoresis direction indicated is from anode to cathode.



We have previously identified Ser as the major site in the solubilized rat adipocyte cGI PDE phosphorylated by protein kinase A in vitro(18) . Ser is located within one of the three consensus sequences for phosphorylation by protein kinase A (-RRXS-) in the rat adipocyte cGI PDE (17) (Fig. 1). This serine was not, however, phosphorylated in cGI PDE in intact adipocytes during isoproterenol stimulation (Fig. 2). Fig. 3(A and B) is a representative example of two-dimensional tryptic phosphopeptide maps of isolated, solubilized adipocyte cGI PDE and rcGI PDE, maximally phosphorylated by protein kinase A to an estimated total phosphorylation stoichiometry of 0.9 ± 0.2 (mean ± S.E., n = 5) (cf. (18) ). When compared to the two-dimensional tryptic phosphopeptide map from [P]cGI PDE phosphorylated in intact adipocytes (Fig. 2B), two major in vitro phosphopeptides tPP5 and tPP6 (Fig. 3, A and B) clearly migrated differently from the tPP. tPP5 and tPP6 were identified as tPP and tPP (Fig. 1) by two approaches. (i) After elution from the TLC plates, tPP5 and tPP6 cross-reacted with anti-P as judged from Tricine-SDS/urea-PAGE of immunopellets and immunosupernatants (Fig. 3C, lanes 5 (tPP) and lane 6 (tPP)). Minor phosphopeptides (denoted tPP1-tPP4) did not react (Fig. 3C, lanes 1, 2, and 4). (ii) tPP5 and tPP6 co-migrated with tPP and tPP respectively, as judged from mixing experiments in which the respective phosphopeptide was eluted from the TLC plate, mixed, and re-run together using two-dimensional phosphopeptide mapping (data not shown). tPP5 (tPP) and tPP6 (tPP) were previously shown to be generated from P by tryptic cleavage C-terminal to Arg or Arg and Arg, respectively(18) . Thus, tPP was clearly distinct from tPP or tPP, i.e. the major site phosphorylated in cells by isoproterenol was different from that phosphorylated in vitro by protein kinase A (Ser).


Figure 3: Two-dimensional tryptic phosphopeptide maps of solubilized cGI PDE phosphorylated in vitro by protein kinase A. Rat adipocyte cGI PDE (A) and rcGI PDE (B) were phosphorylated with protein kinase A in the presence of [-P]ATP. Tryptic phosphopeptides were analyzed by two-dimensional TLE/TLC. C, major tryptic phosphopeptides from protein kinase A-phosphorylated rcGI PDE (tPP1-tPP6), indicated as 1-6, were eluted from the TLC plates and incubated with anti-P. Immunosupernatants and immunopellets were then subjected to Tricine-SDS/urea-PAGE. Lanes 1-6 represent tPP1-tPP6. X marks origin.



One of the minor phosphopeptides (tPP1) (Fig. 3) appeared to migrate similarly to tPP. Eluted tPP1, derived from solubilized rcGI PDE phosphorylated with protein kinase A in vitro, co-migrated with tPP in mixing experiments (data not illustrated). Thus, although most of the protein kinase A-catalyzed phosphorylation of solubilized authentic adipocyte cGI PDE took place on Ser as reported previously(18) , with an estimated phosphorylation stoichiometry of 0.4 ± 0.04 (mean ± S.E., n = 5), some also occurred in the same tryptic peptide as that phosphorylated in intact cells during isoproterenol stimulation (estimated in vitro phosphorylation stoichiometry 0.2 ± 0.04 (mean ± S.E., n = 7)). tPP1 was therefore used to further identify the site phosphorylated in cells, as described below.

The minor tryptic phosphopeptides tPP2, tPP3, and tPP4 migrated differently from the major tryptic phosphopeptide derived from cGI PDE phosphorylated in cells. The presence of tPP3 was variable, being absent in some experiments. The positions of these tryptic phosphopeptides in the enzyme were not identified.

Identification of Tryptic and Asp-N Protease-derived Phosphopeptides from cGI PDE Phosphorylated in Adipocytes

Since the amount of phosphopeptides that could be isolated from cGI PDE phosphorylated in adipocytes was far below that required for amino acid sequencing, tryptic phosphopeptides derived from adipocyte cGI PDE were instead evaluated for co-migration in several separation systems with the tryptic phosphopeptides obtained from protein kinase A-phosphorylated synthetic cGI PDE peptides (Fig. 1). This approach has been used previously in our laboratories for identification of the site of protein kinase A-catalyzed phosphorylation in vitro(18) . As shown in Fig. 4, tPP co-migrated with tPP in the two-dimensional separation system, whereas this was not the case with any of the tryptic cleavage products from PP (data not shown). Thus, the serine phosphorylated in cells during stimulation with isoproterenol appeared to reside in the cGI PDE amino acid sequence Arg-Arg.


Figure 4: Comparison of tryptic phosphopeptides from rat adipocyte cGI PDE phosphorylated in cells with tPP. P was phosphorylated by protein kinase A, trypsinized, and subjected to two-dimensional TLE/TLC (A). Major tryptic phosphopeptides tPP, tPP, and tPP derived from adipocyte cGI PDE phosphorylated in cells were eluted from the TLC plates and re-run without (B-D) or with (E-G) eluted tPP (A). X marks origin.



As shown in Fig. 4, tPP and tPP, which migrated very similarly to tPP in the two-dimensional phosphopeptide mapping system (Fig. 2, B-D), co-migrated with tPP. Thus, stimulation of adipocytes with isoproterenol, insulin, or the combination of both appeared to result in phosphorylation of cGI PDE within the amino acid sequence Arg-Arg. To verify this, the tryptic phosphopeptides were cleaved once more with Asp-N protease (known to cleave N-terminal to cysteic acid or aspartic acid). As shown in Fig. 5, this treatment generated one new phosphopeptide, which in each case again co-migrated with the one obtained from Asp-N protease-treated tPP.


Figure 5: Comparison of Asp-N protease-treated tryptic phosphopeptides from cGI PDE phosphorylated in rat adipocytes with Asp-N protease-digested tPP. tPP and tryptic phosphopeptides from cGI PDE phosphorylated in cells stimulated with isoproterenol, insulin, or both hormones were prepared and subjected to two-dimensional TLE/TLC. Major tryptic phosphopeptides, corresponding to tPP, tPP, tPP, and tPP, were eluted from the TLC plates, re-run (A, D, G, and J), or digested with Asp-N protease and subjected to two-dimensional TLE/TLC as indicated (B, E, H, and K). These Asp-N protease-generated tryptic phosphopeptides from adipocytes (E, H, and K) were eluted and mixed with eluted Asp-N protease-digested tPP (B) and subjected to two-dimensional TLE/TLC as indicated (F, I, and L). The generation of a new phosphopeptide is illustrated by mixing of Asp-N protease-digested and undigested tPP (C). X marks origin.



Identification by Other Methods of the Tryptic Phosphopeptides from cGI PDE Phosphorylated in Adipocytes

tPP1 co-migrated with tPP (see above) and tPP (data not shown). Since tPP1 could be obtained in a sufficiently large amount from rcGI PDE phosphorylated in vitro by protein kinase A, which was not the case with the phosphopeptides derived from cGI PDE phosphorylated in adipocytes, tPP1 was used to verify further the identity of tPP, tPP, and tPP using other separation systems. Using Mono Q, reversed phase C8 chromatography, or Tricine-SDS/urea-PAGE, tPP1 was shown in each case to co-fractionate with tPP (Fig. 6). These results, taken together, demonstrate that the cGI PDE amino acid sequence Arg-Arg contains the serine phosphorylated in situ during incubation with the different hormones.


Figure 6: Separation of tryptic phosphopeptides by Mono Q, C8 reversed phase chromatography, or Tricine-SDS/urea-PAGE. rcGI PDE and P were phosphorylated in vitro by protein kinase A in the presence of [-P]ATP. Tryptic phosphopeptides were separated by two-dimensional TLE/TLC. tPP1 (times), derived from protein kinase A-phosphorylated rcGI PDE and tPP (circle), were eluted from the TLC plates and subjected to chromatography on a Mono Q HR 5/5 (Pharmacia) column (A) or on a reversed phase C8 column (Kromasil C8 5µ, 4.6 times 150 mm) (B), either separately or in combination (bullet). The Mono Q column was equilibrated in 10 mM Tris-HCl, pH 8.0, and eluted with a linear gradient of 0-500 mM NaCl (1 ml/min). The C8 reversed phase column was equilibrated in 0.1% trifluoroacetic acid and eluted with a linear gradient of 0-60% acetonitrile (0.5 ml/min). The elution-position of the P-labeled peptides were detected by liquid scintillation counting. C, tPP1 from protein kinase A-phosphorylated rcGI PDE and tPP were eluted from the TLC plates and subjected to Tricine-SDS/urea-PAGE.



Identification of the Serine Residue in cGI PDE Phosphorylated in Response to Hormone Stimulation of Adipocytes

Two serines, Ser and Ser, are located in the cGI PDE sequence Arg-Arg. To identify the specific phosphorylation site, tPP, tPP, and tPP were coupled to a Sequelon-AA membrane and subjected to radiosequencing. In each case the radioactivity released was found in cycle 3 (Fig. 7). Similar results were found when tPP1 was analyzed (data not shown). This clearly demonstrates that Ser was the site phosphorylated in cGI PDE during incubation of adipocytes with insulin or isoproterenol alone or in combination.


Figure 7: Radiosequencing of tryptic phosphopeptides obtained from cGI PDE phosphorylated in adipocytes. Tryptic phosphopeptides from solubilized cGI PDE from P-labeled rat adipocytes stimulated with isoproterenol and/or insulin were analyzed as described under ``Experimental Procedures'' and in Fig. 2. The major phosphopeptides (tPP, tPP, and tPP) were eluted and coupled to Sequelon-AA membranes and sequenced on an Applied Biosystems gas phase sequencer (model 470A). Released phenylthiohydantoin amino acid-derivatives from each cycle were spotted on TLC plates, and the radioactivity was quantified by digital imaging and volume integration of each spot after subtraction of background P.




DISCUSSION

Using several experimental approaches and fractionation procedures, we have demonstrated that stimulation of adipocytes with insulin, isoproterenol, or the combination of both results in phosphorylation of cGI PDE at a single site (Ser) and activation of the enzyme. Ser is localized within the sequence MFRRPSLPCISREQ, one of three consensus sequences (-RRXS-) for putative protein kinase A-catalyzed phosphorylation of rat adipocyte cGI PDE (Fig. 1). The insulin-induced phosphorylation was presumably mediated by the previously described (14, 15) insulin-stimulated cGI PDE kinase activity found in cytosolic extracts of rat adipocytes following their stimulation by insulin. For technical reasons, it has not yet been possible to identify the serine(s) in cGI PDE phosphorylated in vitro during incubation with such extracts from insulin-treated adipocytes. Several insulin-stimulated serine protein kinases, which appear to have one or several properties in common with insulin-stimulated cGI PDE kinase (e.g. activation by serine/threonine phosphorylation that is blocked by wortmannin treatment of cells), have been found in adipose tissue(28, 29, 30) .

Protein kinase A is likely to be responsible for the isoproterenol-induced phosphorylation of cGI PDE in intact cells. In cell-free preparations, Ser rather than Ser was found, as described previously(18) , to be the major target for protein kinase A-catalyzed phosphorylation of cGI PDE. Ser was found to be phosphorylated by protein kinase A in vitro, less extensively than Ser but to the same extent as in intact cells in response to isoproterenol or insulin (phosphorylation stoichiometry approximately 0.2). This, and the fact that membrane-associated adipocyte cGI PDE has been reported to become activated(31) , and platelet and hepatocyte cGI PDEs phosphorylated and activated in vitro by protein kinase A (32, 33, 34) , indicates that this kinase directly (and not through another kinase) phosphorylates and activates the enzyme in intact cells, although this still remains to be definitely established. The reason for the preferential phosphorylation of Ser rather than Ser by protein kinase A in vitro is not known. However, adipocyte cGI PDE is membrane-bound and in fat cell extracts requires salt and detergent for efficient solubilization. The cDNA structure of the enzyme predicts it to be an intrinsic membrane protein with six putative transmembrane sequences in its N-terminal region(17) . In spite of several attempts with intact and solubilized membrane preparations of cGI PDE, we have so far been unable to establish conditions during which Ser is not extensively phosphorylated by protein kinase A in vitro. (^3)This may indicate that correct membrane insertion of the enzyme is a critical determinant for exposing Ser and not Ser for phosphorylation.

Previous work showing different time-courses for phosphorylation and activation of cGI PDE in rat adipocytes stimulated with isoproterenol or insulin (12, 13) and more than additive phosphorylation/activation during stimulation with both hormones in combination(13, 16) , was tentatively interpreted as indicating phosphorylation at two sites in cGI PDE(10) . Clearly, the present result is not consistent with this hypothesis and instead indicates that the signal pathways interact upstream of cGI PDE. Since the insulin-stimulated cGI PDE kinase appears to be activated in fat cells via phosphorylation during insulin stimulation ((10) ; cf.(35) ), this kinase could be dually regulated by an insulin-stimulated kinase kinase (or an insulin-inactivated phosphatase) and protein kinase A, respectively. However, phosphatidylinositol 3-kinase (36) and insulin receptor substrate-1 (and related proteins)(1, 37) , known upstream links in the antilipolytic signal chain, could also be merging points. cAMP-enhancing agents have recently been reported to modulate the signals from insulin and other growth factors(38, 39, 40, 41, 42, 43) , generally by attenuating them, but at least in one case by enhancing the effect of growth factors(43) , consistent with the findings in this report. The identification reported here of the site in cGI PDE phosphorylated in cells in response to both insulin and cAMP-enhancing agents should be useful for the further dissection of the signal transduction pathway, linking the hormone receptor events to the activation of cGI PDE in the control of adipose tissue lipolysis.


FOOTNOTES

*
This work was supported by grants from the following foundations: A. Påhlsson, Malmö, Sweden; Dr. P. Håkansson, Eslöv, Sweden; Lars Hierta, Stockholm, Sweden; Crafoord, Lund, Sweden; Swedish Diabetes Association, Solna, Sweden; Nordisk Insulin, Copenhagen, Denmark; Faculty of Medicine, Lund University; and Swedish Medical Research Council Project 3362. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Section for Molecular Signalling, Lund University, P. O. Box 94, S-221 00 Lund, Sweden. Tel.: 46-46-228587; Fax: 46-46-2224022.

(^1)
The abbreviations used are: cGI PDE, cGMP-inhibited phosphodiesterase; PDE, cyclic nucleotide phosphodiesterase; rcGI PDE, recombinant NIH-3006 fibroblast cGI PDE; HAc, glacial acetic acid; PAGE, polyacrylamide gel electrophoresis; TLE, thin layer electrophoresis; TLC, thin layer chromatography; tPP, tryptic phosphopeptide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^2)
M.-J. Leroy, E. Degerman, M. Taira, L. H. Wang, M. Movsesian, E. Meacci, and V. C. Manganiello, submitted for publication.

(^3)
S. Resjö, T. Rahn, H. Tornqvist, V. C. Manganiello, P. Belfrage, and E. Degerman, unpublished results.


ACKNOWLEDGEMENTS

Gunilla Henningsson and Ann-Kristin Holmén Pålbrink provided valuable technical assistance. We thank Dr. Martha Vaughan for critical reading of this manuscript.


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