identification of the phosphorylation site in vitro for camp-dependent

THE JOURNAL OF BIOLCGICAL CHEMISTRY Vol. 269, No. 16, Issue of April 22. pp. 11962-11966, 1994 Printed in U.S.A.

Identification of the Phosphorylation Site in Vitro for CAMP-dependent Protein Kinase on the Rat Adipocyte cGMP-inhibited C A M P Phosphodiesterase*

(Received for publication, December 9, 1993)

Ana Rasc6nS9, Eva DegermanS, Masato Tairan, Elisabetta Meaccin, Carolyn J. Smith$ Vincent ManganielloR Per Belfraget, and Hans "nqvistll From the $Department of Medical and Physiological Chemistry and IPaediatrics, University of Lund, Sweden and the Wboratory of Cellular Metabolism. National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Rat adipocyte cGMP-inhibited CAMP phosphodiesterase (cGI-PDE) appears to be dually regulated in intact cells by serine phosphorylations induced by isoprenaline and insulin, respectively (Degerman, E., Smith, C. J., 'Ibrnqvist, €I., Vasta, V., Belfrage, P., and Manganiello, V. C. (1990) Proc. NatZ. Acad. Sci. U. S. A. 87,533637; Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manga- niello, V. C. (1991) J. Biol. Chem. 266,13386-13390). Since CAMP-dependent protein kinase (CAMP-PK) catalyzes the p-adrenergic effects, the site in the isolated cGI-PDE phosphorylated by this kinase was explored. A peptide, LRRSSGASGUTSEHHSR (P18), corresponding to the amino acid sequence Leum-Arp in the putative regulatory domain of the rat adipocyte cGI-PDE was synthesized. It contains a consensus substrate sequence -RRxs- for CAMP-PK within two tryptic cleavage sites and was readily phosphorylated by CAMP-PK. ' h o phosphopeptides, identified as RS-[SaPISGASGLLTSEHHSR and S-[88PJSGASGLLTSEHHSR, were obtained after stoichiometric phosphorylation and trypsinization of the peptide. These two peptides and the two main tryptic phosphopeptides obtained from immunoisolated [SaP]cGI-PDE phosphorylated with CAMP-PK in a solubilized crude adipocyte membrane fraction were immunoprecipitated by an affinity-purified polyclonal antibody raised against P18 and exhibited the same chromatographic and electrophoretic profiles in three different separation systems. Similar radiosequencing profiles indicated that the second most N-terminal serine, corresponding to Ser-427 in the intact cGI-PDE, was phosphorylated by CAMP-PK in both Pl8 and authentic cGI-PDE. It is concluded that serine 427 is the target for cANLP-PK phosphorylation of the rat adipocyte cGI-PDE in vitro.

Stimulation of adipocytes with insulin or agents that increase intracellular CAMP results in activation of the membrane-associated cGMP-inhibited CAMP phosphodiesterase (cGI-PDE)' (1). The enzyme, with a subunit M, of 135,000 by

* This work was supported by grants from A. Phhlssons, Malmo; the Novo Nordisk Foundation, Copenhagen; the Swedish Diabetes Associa- tion; the Swedish Hoechst, Stockholm; the Lars Hierta, Stockholm; the Medical Faculty, University of Lune and Swedish Medical Research Council Project numbers 3362 and 8689. The costs of publication of this article were defrayed in part by the payment of page charges. This

with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked "aduertisement" in accordance

8 Present address: Escuela de Biologia, Universidad Central de Ven- ezuela, Apartado 47069, Caracas 1041, Venezuela.

The abbreviations used are: cGI-PDE, cGMP-inhibited CAMP phosphodiesterase; cAMP-PDE, cAMP phosphodiesterase; CAMP-PK, CAMP-dependent protein kinase; C,,El,, heterogeneous nonionic alkyl

SDS-PAGE, has been purified from rat adipose tissue and further characterized (2, 3). Recently, human cardiac (4) and rat adipocyte (5) cGI-PDE full-length cDNAs have been cloned and sequenced. The deduced primary structures of these distinct cGI-PDEs predict a C-terminal catalytic domain con- served among all PDE families and a nonconserved N-terminal catalytic region that is thought to contain regulatory domains, e.g. phosphorylation sites and sites participating in allosteric interactions (6). We recently showed that stimulation of intact rat adipocytes with isoprenaline or insulin induced serine phosphorylation of cGI-PDE in a concentration- and time-dependent manner, which correlated with activation of the enzyme (7, 8). Exposure of adipocytes to insulin in the presence of isoprenaline produced synergistic activation and phosphorylation of the cGI-PDE under conditions where insulin lowered CAMP-PK activity and lipolysis (8, 9). These results suggest that distinct phosphorylation sites on the cGI-PDE are targets for CAMP-PK and insulin-dependent serine protein kinaseh), respectively.

In the present work we have localized the phosphorylation site for CAMP-PK on the isolated rat adipocyte cGI-PDE. Since identification by direct sequencing of proteolytic peptides from the adipocyte cGI-PDE was not possible due to insufficient amounts of purified enzyme and its susceptibility to nonspecific proteolysis during purification, an alternative strategy was de- veloped to identify the peptide sequence containing the phosphorylation site. Based on the deduced amino acid sequence of the rat adipocyte cGI-PDE (51, a peptide containing the putative CAMP-PK phosphorylation site was synthesized and an antibody was raised against this peptide. After phosphorylation of the synthetic peptide and the adipocyte cGI-PDE by CAMP- PK, the chromatographic and electrophoretic behavior of tryptic phosphopeptides from the two were compared and found to be identical. The exact location of the phosphorylated serine was then assessed by radiosequencing.

EXPERIMENTAL PROCEDURES Materi~Zs-[2,8-~H]CAMP (36.4 Ci/mmolj from DuPont NEN,

Dreilich, Germany, was purified (lo), and [y-'*PlATP was synthesized (11). The catalytic subunit of CAMP-PK from bovine heart with a specific activity of 30-65 unitdmg (1 unit is defined as the transfer of 1 pmol of phosphate from ATP to casein per min at pH 6.5 at 37 " 0 , Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), and rabbit CAMP-PK inhibitor were obtained from Sigma; N-tosyl-L-phenylalanine chloromethyl ketone-

polyoxyethylene glycol detergent (abbreviated as C,E,, from the general formula C,E,+ ,(OCH,CH,j,OH); OPC-3911, (N-cyclohexyl-N-2-hydroxyethyl-4-(6-(1,2-dihydro-2-oxoquinolyloxy~but~amide; Tricine- SDS/urea-PAGE, discontinuous Tricine SDS urea polyacrylamide gel electrophoresis; TPCK-trypsin, N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin; Mops, 3-(N-morpholino)propanesulfonic acid.

11962

CAMP-PK Phosphorylation Site on cGI-PDE 11963 treated trypsin (TPCK-trypsin, sequencing grade) from Worthington; the heterogeneous nonionic alkyl polyoxyethylene glycol detergent C13E12 from Berol Kemi AB, Stenungsund, Sweden. OPC-3911 (N-cyclohexyl-N-2-hydroxyethyl-4-(6-(l,2-dihydro-2-oxoquinolyloxy~butyra- mide) was generously supplied by Prof. H. Hidaka of Nagoya School of Medicine, Japan. The IgG fraction (2 mg/ml) of a specific polyclonal rabbit antiserum, here termed aPDE, which was raised against cGI- PDE purified from bovine adipose tissue and cross-reacted with rat fat cG1-PDE, was used for immunoprecipitation (3) in some experiments.

Synthesis of cGI-PDE Peptides and Preparation of Antibody against the Peptide P18-The adipocyte cGI-PDE peptides, LmGASGLLT- SEHHSR (P18; residues 423-440 in Taira et al. (5)) and L m G A ( P 7 ; residues 423-429), containing the putative CAMP-PK phosphorylation site, as well as the corresponding human cardiac cGI-PDE peptide, GLLWSTWITTTSATGLPTLEPADVR (P24), were synthesized on an automated solid-phase peptide synthesizer (Applied Biosystems model 430A) using the Fmoc (N49-fluoreny1)methoxycarbonyl) program provided by the manufacturer and purified by preparative reversed- phase chromatography (Kromasil C8 5 p, 20 x 250 mm) to 91 and 97% purity, respectively. The identity of each peptide was verified by mass spectrometry (BioIon 20, Applied Biosystems) and amino acid analysis. A polyclonal rabbit antibody, aP18, was raised against the P18 peptide coupled to keyhole limpet hemocyanin and purified over a column with the peptide coupled to CNBr-Sepharose 4B.

Phosphorylation of the Synthetic cGI-PDE Peptide-For maximal phosphorylation, each peptide (25 nmol) was incubated at 30 "C for 1 h with 10 units of the catalytic subunit of CAMP-PK in 200 rJ of 20 Mops, 0.2 m~ dithioerythritol, 16 n" magnesium acetate with 1 m~ [-y-32P]ATP (100-500 dpdpmol) or unlabeled ATP. The phosphorylated peptide was applied to a C18 disposable minicolumn (Chromabond) in 0.1% trifluoroacetic acid, washed extensively with the same buffer to remove [y3'PlATP, and eluted in 75% acetonitrile, 0.1% trifluoroacetic acid. For kinetic experiments, phosphorylation of the respective synthetic peptide was assayed in a final volume of 50 pl of 200 p~ IyS2PIATP (100-200 dpm/pmol), 20 nm Mops, 10 m~ magnesium acetate with AMP-PK, 1.25 pg/ml, at pH 7.0. After incubation for 5 min at 30 "C, duplicate samples (10 pl) were spotted on Whatman P81 phosphocellulose paper, washed five times in 75 m~ phosphoric acid and once with acetone, and dried before radioassay by liquid scintillation counting.

Generation and Analysis of cGI-PDE Pyptic 32P-Phosphopeptides from Intact Rat Adipoqte cGI-PDE Phosphorylated by dMP-PK- Adipocytes from epididymal fat pads of 150-g Sprague-Dawley rats (ALAB, Stockholm, Sweden) fasted overnight were prepared (12) in buffer containing 200 rm adenosine and 2 nm glucose. Adipocytes from 10 rats were washed twice with 10 volumes of 20 m~ Tris, pH 7.4/1 m~ EDTN255 m~ sucroselleupeptin, antipain, and pepstatin, each 10 pg/ ml, homogenized at room temperature, immediately cooled to 4 "C, and centrifuged (100,000 x g, 60 min, 4 "C). Homogenization in low ionic strength buffer at room temperature was essential to avoid loss of cG1-PDE into the floating fat cake. The microsomal cell pellet, containing >80% of total homogenate cG1-PDE activity, was solubilized in 1 ml of 50 m~ Tris, pH 7.6,5 m~ MgCl,, 1 nm EDTA, 0.1 m~ EGTA, 100 n" NaBr, 50 n" NaF, 1% (v/v) CI3El2, 3 m~ benzamidine, 10 pg/ml leupeptin, 5 pg/ml antipain, 1 pg/ml pepstatin using a glass homogenizer. After 60 min on ice and centrifugation (10,000 x g, 30 min, 4 "C), the super- natant, containing >SO% of original pellet CAMP-PDE activity, was di- luted with an equal volume of the same buffer lacking NaBr, NaF, and C13E,, but containing 40% (w/v) glycerol, and stored at -80 "C. cGI- PDE accounted for more than 95% of the CAMP-PDE activity in the solubilized fraction. High affinity CAMP-PDE activity was assayed with 0.5 p~ 13HlcAMP, and cGI-PDE activity was defined as the fraction of that activity inhibited by 3 p~ OPC 3911 (2).

The detergent-solubilized microsomal fraction (approximately 30 pg of total protein) containing 20 pmol/min cG1-PDE activity, which was calculated to correspond to -3.1 ng of pure enzyme protein (based on specific activity, 5.4 mol/min/mg of protein, of purified cGI-PDE(2)) was incubated at 30 "C for 20 min with CAMP-PK catalytic subunit, 20-200 unitdml, in a final volume of 0.9 ml of 30 m~ Hepes, pH 7.4,lO m~ EDTA, 0.1 m~ EGTA, 2 n" dithiothreitol, 8 n" MgCl,, 0.5 p~ CAMP, 3 n" benzamidine with leupeptin and pepstatin, each 10 pg/ml, and 40 w [r3ZPlATP (20,000 cpdpmol). The reaction was stopped by adding 90 p1 of a solution of 32 m~ ATP, 0.1 M EDTA, pH 7.5, 30 p~ protein kinase inhibitor and transferred to ice.

The phosphorylation mixture was incubated for 12 h at 4 "C with 20 pl of nPDE followed by immunoprecipitation for 30 min with 80 pl ofa 10% Slurry of Staphylococcus aureus-bound protein A, prewashed to reduce nonspecific interactions (13). The immunoprecipitate was

washed five times in 10 m~ phosphate buffer, pH 7.3, containing 135 nm NaC1, 3 nm KC1, and 0.1% N-lauryl sarcosine, boiled in SDS-PAGE sample buffer, and subjected to electrophoresis in an 8% SDS-PAGE slab gel (14). The gel was frozen and autoradiographed (Kodak X-OMAT-AR), and the 135-kDa cGI-PDE band was identified and excised. The stoichiometry of cGI-PDE phosphorylation was estimated as moles of 32Pi incorporated in the 135-kDa gel band per mol of cGI-PDE protein applied, based on enzyme activity measurement in the solubilized membrane preparation and the specific activity of pure cGI-PDE.

The 135-kDa cG1-PDE polypeptide was eluted from excised gel pieces (15). Recovery of [32PlcGI-PDE through this procedure was 75 f 8% (mean 3 S.D., n = 5). Trypsinization was then performed in 50 pl of 100 m~ N-ethylmorpholine acetate buffer, pH 8.0, for 2 h at 37 "C with 10 pg of TPCK-trypsin. Reaction was stopped by adding 1 pl of 0.1 M diiso- propylfluorophosphate and 400 pl of h0. The 32P-phosphopeptides that were generated were recovered in the soluble fraction.

Analytical separations of tryptic 32P-phosphopeptides from cGI-PDE or the synthetic peptide was performed by: 1) discontinuous Tricine- SDSIurea-polyacrylamide gel electrophoresis (Tricine-SDS/urea-PAGE) (16) using 16.5% T, 6% C, and 6 M urea in the lower separating gel; 2) anion exchange chromatography on a Pharmacia Mono Q column (HR 5/5), with a linear 0-0.5 M NaCl gradient in 10 n" Tris-HC1, pH 8.0, at a flow rate of 0.5 mumin; 3) reversed-phase HPLC on a C8 column (Kromasil C8 5 p, 4.6 x 150 mm) with a linear 0-75% acetonitrile gradient in 0.1% trifluoroacetic acid at a flow rate of 0.5 mumin. When comparing elution positions of tryptic 32P-phosphopeptides from cGI- PDE with those from the synthetic peptide, the samples, adjusted to identical volumes and buffer composition, were run on computerized high performance liquid chromatography equipment (Kontron Instru- ments) en suite using the same buffers to minimize between-run differ- ences.

Isolated 32P-phosphopeptides were sequenced using an Applied Bio- system Protein Sequencer 477Aequipped with PTH Analyzer 12OA. The phosphorylation site(s) within a phosphopeptide were located by moni- toring the release of 32Pi from the phosphopeptide at each cycle of Ed- man degradation by thin layer electrophoresis (17).

Immunoprecipitation of Pyptic 3ZP-Phosphopeptides-Tryptic digests containing 32P-phosphopeptides were incubated with aP18 for 16 h at 4 "C; the immunoprecipitates were collected with S. aureus-bound protein A and analyzed by Tricine-SDWurea-PAGE. The antibody precipitated 32P-P18 and trypsinized 32P-P18 but not 32p-P24, 32P-P7, or 32P-Kemptide (data not shown).

RESULTS Phosphorylation and Characterization of the Synthetic Pep-

tide Derived from cGZ-PDE-The recent cloning and sequencing of the rat fat cGI-PDE (5) made it possible to identify several potential sites for phosphorylation by CAMP-PK based on the consensus sequence -RRXS- (18) in the putative regulatory domain of the enzyme (Table I). In preliminary experiments with cGI-PDE phosphorylated in vitro by CAMP-PK, the tryptic phosphopeptides bound to an anion exchange column at pH 8.0 and to a cationic exchange column at pH 5.0 consistent with the presence of histidine. This ruled out the first two peptides (Table I) because of their charge properties. The third and histidine-containing peptide sequence (Table I) was con- sidered the most likely candidate for the CAMP-PK phosphorylation site. The size of the synthetic peptide was chosen so that following phosphorylation by cAMP-PK tryptic digestion would generate the same products as those generated from

This peptide LmGASGLLTSEHHSR was phosphorylated by CAMP-PK to the extent of ~0.7 mol of phosphate/mol of peptide. K,,, and V,, values for this peptide, its shorter version L m G A (P7), and Kemptide (19) were similar (Table 11).

Trypsinization of phosphorylated P18 (trypsin:peptide, 1:100, mollmol) for 30 min at 37 "C resulted in the generation of two phosphopeptides (Fig. 1B, lane 1 ), as expected (15), due to the presence of 2 arginine residues in tandem closely followed by a phosphorylated serine. These two peptides were separated on a Mono Q anion exchange column (Fig. 2A). The first peak ( p l ) corresponds to the low mobility phosphopeptide seen on Tricine-SDWurea-PAGE and the second peak ( p 2 ) to

cGI-PDE.

11964 CAMP-PK Phosphorylation Site on cGI-PDE TABLE I

Putative cAMP-PKphosphorylation sites in the deduced amino acid sequence of rat adipocyte cGI-PDE Consensus sequences for phosphorylation by CAMP-PK are underlined.

phosphorylated try tic peptides Calculated net charge" of

pH 5.0 pH 8.0

Peptide sequence ( R x s - , X L h

1. -mCVSLGESAAGYYGSGK-(residues 277-296) 2. -mLPCISR-(residues 299-308) 3. -mGASGLLTSEHHSR-(residues 424-440)

-0, -1 +1, 0 +2, +1

-1, -2 0, -1

-1, -2 Assuming that phosphoserine has a charge of -2 at pH 8.0 and -1 at pH 5.0 (14).

* Each sequence yields two different peptides upon trypsinization.

TABLE I1 Kinetic data for cAMP-PK-induced phosphorylation of Kemptide and

rat adipocyte cGI-PDE-derived synthetic peptides Peptide phosphorylation was performed as described under "Experi-

menatl Procedures" with 1.25 pg/ml of the CAMP-PK catalytic subunit. Data are means of values from three separate experiments in which values varied less than 10%.

Peptide Km (*PPI "mal P M pmollminlmg

LRRASLG (Kemptide) 15 LRRSSGASGLLTSEHHSR 30

1.4 2.0

LRRSSGA 40 1.1

A .*

kDa *

- 180

135 - 116

- 84

- -front

L

1 2 FIG. 1. A, SDS-PAGE of adipocyte cG1-PDE from a crude membrane

fraction phosphorylated with CAMP-PK and immunoprecipitated with anti-cGI-PDE IgG (cf. "Experimental Procedures"). B, separation using Mcine-SDShea-PAGE of phosphopeptides in tryptic digests of the excised 135-kDa [32PlcGI-PDE polypeptide (lane 2 ) and of the synthetic 32P-phosphopeptide (lane 1 ).

the fast moving one (Fig. 2A, inset). A small third peak of radioactivity contained traces of nonspecifically proteolyzed peptide seen only after extensive trypsinization (data not shown). N-terminal sequencing identified them as RSSGAS- GLLTSEHHSR (pl) and SSGASGLLTSEHHSR (p2), respectively. The second most N-terminal serine was shown to be phosphorylated since all 32Pi was released from p l at the third cycle of Edman degradation, and at the second cycle from p2 (Fig. 4, A and B ) .

The characteristics of these two synthetic tryptic phosphopeptides were then compared with those of tryptic peptides generated from [32P]cGI-PDE to identify the in vitro CAMP-PK phosphorylation site on cGI-PDE.

Phosphorylation of Adipocyte cGZ-PDE-The catalytic subunit of CAMP-PK (120 unitdml) catalyzed phosphorylation of intact 135-kDa cGI-PDE (Fig. lA) in solubilized crude microsomal membranes from rat adipocytes. Phosphorylation was

maximal in 5-10 min, with an estimated stoichiometry of 0.9 2 0.1 (S.D., n = 6) mol of phosphateImo1 of cGI-PDE 135-kDa polypeptide. None or trace phosphorylation occurred in the ab- sence of CAMP-PK or in the presence of 5 p~ protein kinase inhibitor. The use of several protease inhibitors, immediate immunoisolation on ice, and maintenance of total protein concentration in the phosphorylation mixture below 100 pg/ml were essential to obtain optimal phosphorylation and to minimize proteolytic degradation of the 135-kDa cGI-PDE.

Identification of the CAMP-PK Phosphorylation Site on the cGZ-PDE-Tkypsinization of intact [32P]cGI-PDE generated two predominant 32P-phosphopeptides as analyzed on Tricine-SDS/ urea-PAGE (Fig. lB, lane 2 ) with the same mobilities as the synthetic RS-[32P]SGASGLLTSEHHSR and S-[32P]SGASGLL- TSEHHSR phosphopeptides. Recovery of soluble 32P-phosphopeptides from [32P]cGI-PDE was 75 2 6% (mean 2 S.D., n = 4). The remaining insoluble fraction recovered from the tube using phosphoric acid and boiling in SDS contained no other 32P-phosphopeptide as analyzed by Tricine-SDSIurea-PAGE (not shown). The tryptic digest, to which 3-5 pg of unlabeled phosphorylated and trypsinized synthetic peptide was added as a carrier to increase chromatographic yields, was subjected to anion exchange chromatography (Fig. 2 B ) . Two distinct peaks (pl and p2) were eluted at the same positions as the RS-[32P]- SGASGLLTSEHHSR and S-[32P]SGASGLLTSEHHSR phosphopeptides (cf: Fig. 2 4 ) containing 19 and 47%, respectively, of total 32P-material eluted. Furthermore, p l had the same mobility as the RS-[32P]SGASGLLTSEHHSR phosphopeptide and p2 as the S-[32P]SGASGLLTSEHHSR phosphopeptide on Tricine-SDSIurea-PAGE (Fig. 2, insets). At pH 5.0, both the synthetic and the cGI-PDE-derived tryptic "P-phosphopeptides bound to and eluted at the same position from a cationic column (Mono S, Pharmacia; data not shown). To provide further evidence of their identity, the tryptic digests of [32P]~GI- PDE and synthetic 32P-phosphopeptide were compared on a C8 reversed phase column (Fig. 2, C and D). Again, the elution positions of the [32P]cGI-PDE phosphopeptides were the same as those of the synthetic phosphopeptides.

To verify the identity of these peptides, the affinity-purified polyclonal antibody aP18 raised against the synthetic peptide P18 was tested for recognition of [32PlcGI-PDE andor its tryptic phosphopeptides. aP18 could detect 300 pg of cGI-PDE (based on specific activity of the purified enzyme) on a Western blot of a solubilized adipocyte microsomal fraction, as well as selectively immunoprecipitate cGI-PDE from this fraction (Fig. 3A). P18 and its tryptic cleavage products were also quantita- tively immunoprecipitated. P24, a peptide containing the putative CAMP-PK motif from human cardiac cGI-PDE, P7, or Kemptide could not be immunoprecipitated with aP18 (not shown). As shown in Fig. 3B, aP18 immunoprecipitated the same two tryptic 32P-phosphopeptides generated from SDS- PAGE-isolated [32P]cGI-PDE as well as from a trypsinized adipocyte microsomal fraction phosphorylated with CAMP-PK. Unlabeled P18, but not P24, blocked this immunoprecipitation.

CAMP-PK Phosphorylation Site on cGI-PDE 11965

?i20EAo.5 0 - 'y

10 0.25 6 a m 0

P

- a Y

N

0 0 0 10 20 30 40

pl p2 tryplic digest

B

P - " - cG" PDE I ..

Elution time min

200 , 0.5 , I

Elution lime min

- 0 10 20 30 40 50

0 10 20 30 40 50

Elution lime min

FIG. 2. Chromatographic separation of tryptic 32p-phosphopeptides. Tryptic digests (cf. "Experimental Procedures") of the synthetic peptide LRRSSGASGLLTSEHHSR (A) and adipocyte cGI- PDE (B) , 3'P-phosphorylated by CAMP-PK, were separated on a Mono Q HR5/5 (Pharmacia) column equilibrated in 10 m Tris-HC1, pH 8.0, with a 0-0.5 M linear NaCl gradient at 0.5 mumin. An aliquot from each peak was then separated using Tricine-SDS/urea-PAGE for identification (insets). Recoveries of material applied were 93% (A) and 80% ( B ) , respectively. Tryptic digests of M P - P K 32P-phosphorylated synthetic peptide (C) and cGI-PDE (D) were cleaned over a disposable 100-mg C18 minicolumn (Chromabond ec) and separated on a C8 reversed phase column (Kromasil C8 5 p, 4.6 x 150 mm) in 0.1% trifluoroacetic acid and a linear 0-75% acetonitrile gradient a t a flow rate of 0.5 mVmin. Recoveries of material applied were 78 and 93%, respectively.

In conclusion, this antibody selectively reacts with an epitope on cGI-PDE within residues Le~~'~-Are' and identifies the CAMP-PK phosphorylation site within this sequence.

Finally, the tryptic 32P-phosphopeptides from cGI-PDE were subjected to radiosequencing to reveal the exact location of the phosphoserine residue. From the mixture of unfractionated tryptic peptides released from [32P]cGI-PDE containing RSS- GASGLLTSEHHSR and SSGASGLLTSEHHSR, all inorganic 32Pi was released in cycles 2 and 3 (data not illustrated). After anion exchange chromatography of the cGI-PDE tryptic pep-

1 2 3 1 2 3 4 5 6 7

FIG. 3. Immunoidentification of cGI-PDE and ita tryptic s2p- phosphopeptides with PI8 antipeptide antibody. A, Western blot analysis of cGI-PDE in crude adipocyte microsomal fraction (38 pg of proteidlane containing -3.8 ng of cGI-PDE): lane 1, Coomassie stain; lane 2, Western blot with crP18; lane 3, immunoprecipitation with aP18 followed by Western blot with aP18. B, immunoprecipitations of tryptic 32P-phosphopeptides obtained from cGI-PDE eluted from SDS-PAGE gel (lanes 1 and 2 ) or obtained from the entire solubilized microsomal fraction (lunes 4-7) phosphorylated with CAMP-PK. Immunoprecipita- tion with aP18 alone (lanes 1 and 4 ) , in the presence of excess P18 (lane 5) or P24 (lane 6) peptide, or with preimmune serum (lanes 2 and 7) . Lane 3, P18 peptide phosphorylated with CAMP-PK and trypsinized.

tides (cf Fig. 2B ), sequencing of phosphopeptide p l (RSSGAS- GLLTSEHHSR) released all of the 32Pi at cycle 3 (Fig. 4C). These findings are consistent with the conclusion that the second of the two vicinal serines is the site of phosphorylation, in agreement with the results from corresponding synthetic tryptic peptides (Fig. 4,A and B ). Thus, serine 427 appears to be the target for CAMP-PK phosphorylation of the adipocyte cGI-PDE in vitro.

DISCUSSION Serine 427 in rat adipocyte cGI-PDE was identified as the

site phosphorylated in vitro by CAMP-PK. Properties of the tryptic phosphopeptides from the 135-kDa [32P]cGI-PDE subunit were compared with those of the phosphorylated tryptic cleavage products, RSSGASGLLTSEHHSR and SSGASGLLT- SEHHSR, of the peptide LRRSSGASGLLTSEHHSR. This peptide had been synthesized based on the deduced sequence Leu423-Arg440 in the putative regulatory domain of the recently cloned rat adipocyte cGI-PDE cDNA(5). I t contains a consensus sequence -RRSS- for CAMP-PK and was phosphorylated with essentially the same K, for ATP and V,, as that of the stan- dard CAMP-PK substrate Kemptide. Tryptic cleavage products from 135-kDa cGI-PDE and the synthetic peptide phosphorylated with [y32P]ATP and CAMP-PK exhibited identical electrophoretic mobilities and chromatographic profiles during Tricine-SDSIurea-PAGE and anion exchange and reversed phase chromatography. Furthermore, an antibody raised against the synthetic peptide specifically recognized these tryptic phosphopeptides as well as intact cGI-PDE. Release of 32Pi during sequencing indicated that the same serine site, Ser427, was phosphorylated in the synthetic peptides and in the peptides generated from the [32PlcGI-PDE. Identification of the phosphorylation site by direct sequencing will most likely not be possible until sufficient cGI-PDE can be produced as a re- combinant protein. Studies with intact cells stimulated with isoprenaline will be required to prove that the same serine residue is also the target in situ.

cGI-PDE is likely to be phosphorylated by more than one serine protein kinase since serine phosphorylation seems to

11966 CAMP-PK Phosphorylation Site on cGI-PDE

A

Cycle 1 2 3

+i

3+i I I

Ser Ser Gly

B

1 2 3

C

7+ 1 2 3

Arg Ser Ser Arg Ser Ser FIG. 4. Release of s 2 p i during peptide amino acid microsequencing. 32P-Phosphopeptides were subjected to gas-phase amino acid sequenc-

ing. After each of the first three cleavage cycles, part of the filter disc was removed and 3ZP-products were eluted with 50% (v/v) formic acid and separated on thin-layer electrophoresis a t pH 1.9 to identify release of 32Pi from the phosphopeptideb). A, the SSGASGLLTSEHHSR ( p 2 , Fig. M); B, the RSSGASGLLTSEHHSR ( p l , Fig. M ) synthetic peptide; and C , the RSSGASGLLTSEHHSR ( p l , Fig. 2 B ) tryptic 32P-phosphopeptide material derived from adipocyte cGI-PDE phospho-rylated in vitro with CAMP-PK and separated by Mono Q anion exchange chromatography.

regulate its activity in intact cells in response to both P-adrenergic agents, such as isoprenaline, insulin (71, and growth hormone.2 Results with intact adipocytes are consistent with the notion that isoprenaline and insulin catalyze the phosphorylation of separate sites on cGI-PDE. Both hormones phospho- rylate and activate cGI-PDE with different time courses, and isoprenaline-induced phosphorylation may be more effective in activation of cGI-PDE than that induced by insulin (8). Fur- thermore, simultaneous stimulation of cells with isoprenaline and insulin result in synergistic activation and phosphorylation (8, 9). The identification of the site on cGI-PDE phosphorylated by CAMP-PK in vitro should contribute to the under- standing of the mechanisms of dual hormonal regulation of this enzyme in situ since similar approaches can now be utilized in attempting to identify the site(s) phosphorylated in response to insulin and growth hormone.

Acknowledgments-Zuzana Valnickova, Chatarina Mattsson, and Gunilla Henningsson gave valuable technical assistance. We are grate- ful to Dr. Henry Franzen for the synthesis and purification of the peptides used in this study and Dr. Jacob Donner for the help with the sequence analysis.

E. Eriksson, E. Degerman, and H. Tornqvist, manuscript in preparation.

REFERENCES 1. Manganiello, V. C., Degerman, E., Smith, C. J., Vasta, V., Tornqvist. H., and

2. Degerman, E., Belfrage, P., Newman, A. H., Rice, K. C., and Manganiello, V. C. Belfrage, P. (1992)Adv. Second Messenger Phosphoprotein Res. 25,147-164

3. Degerman, E., Manganiello, V. C., Newman.A. H., Rice, K. C., and Belfrage, P. (1987) J. Bid. Chem. 262,5797-5807

4. Meacci, E., Taira, M., Moos, M., Jr., Smith, C. J., Movsesian, M. A,, Degerman, (1988) Second Messengers Phosphoproteins 12, 171-182

E., Belfrage, P., and Manganiello, V. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,3721-3725

5. Taira, M., Hockman. S. C., Calvo, J. C., Taira, M., Belfrage, P.. and Manga- niello, V. C. (1993) J. B i d . Chem. 268, 18573-18579

6. Conti. M., Jin, S. L. C., Monaco, L., Repaske, D. R., and Swinnen. J. V. (1991) Endocr: Rev. 12,218-234

7. Degerman, E., Smith, C. J., Tornqvist, H., Vasta, V., Belfrage, P., and Manga-

8. Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C.

9. Smith, C. J. , and Manganiello, V. C. (1989) Mol. Pharmacol. 35, 381-386 10. Kincaid, R., and Manganiello, V. C. (1988) Methods Enzymol. 158, 457-471 11. Chang, K. J., Marcus, N. A., and Cuatrecasas, P. (1974) J. B i d . Chem. 249,

6854-6865 12. Honnor, R. C., Dhillon, G. S.. and Landos. C. (1985) J. Biol. Chem. 260,

15122-15129 13. Doolittle, M. H.. Mawbin, D. C., Davis, R. C., Reuben, M. A,, and Elovson, J.

(1991)Anal. Biochem. 195,364368 14. Laemmli, U. K. (1970) Nature 227,680-685 15. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Method9 Enrymol. 201,

16. Schagger, H.. and von Jagow, G. (1987)Anal. Biochem. 166, 368-3379 17. Wang, Y., Fiol, C. J., De Paoli-Roach, A. A., Bell, A. W., Hermodson, M. A,, and

18. Kennely, P. J., and Krebs, E. G. (1991) J. Biol. Chem. 266,15555-15558 19. Kemp, B. E., Graves, D. J., Benjamini, E., and Krebs, E. G . (1977) J. Biol.

niello, V. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 533537

(1991) J. Bid. Chem. 266,13385-13390

110-152

Roach, P. J. (1988) Anal. Biochem. 174,537547

Chem. 260, 15122-15129

identification of the phosphorylation site in vitro for camp-dependent

Documents