THE OF BIOLOGICAL No. 8, of 1980 in Subcellular ... · Photoaffinity labeling with 8-N&*P]cIMP was performed using 25 to 150 pg of protein and incorporation of radioactive label into

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 8, Issue of April 25, pp. 3777-3785. 1980 Printed in U. S. A.

Subcellular Localization of Cyclic GMP-dependent Protein Kinase and Its Substrates in Vascular Smooth Muscle*

(Received for publication, October 17, 1979)

Harlan E. Ives,$ John E. Casnellie,g Paul Greengard, and James D. Jamiesonl From the Section of Cell Biology and the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

The subcellular location of cGMP-dependent protein kinase and its substrates in rabbit aortic medial smooth muscle was studied by subjecting homogenates to a single 4 X lo’ g-min centrifugation on an 8.5 to 60% sucrose density gradient at low ionic strength. Sub- strate proteins GO to GI co-sedimented with a median density of 1.10, while the enzyme (measured by binding of the photoaffinity ligand 8-N3-C3’P]cIMP) was found both at the top of the gradient (with soluble proteins) and in a second peak with median density of 1.12. Gradient fractions were characterized by morphology, assay of the marker enzymes galactosyltransferase, 5‘- AMPase, phosphodiesterase I, NADPH-cytochrome c (P-450) reductase, acid phosphatase, succinate cytochrome c reductase, and determination of RNA, DNA, and protein. Fractions rich in cGMP-dependent protein kinase and its substrates corresponded closely with markers for the plasma membrane, Golgi complex, and endoplasmic (sarcoplasmic) reticulum; electron microscopy indicated that these fractions contained membrane sheets and smooth closed vesicles of various sizes.

To improve the resolution of the smooth membranous organelles, homogenates were pretreated with digitonin prior to centrifugation. The cGMP-dependent protein kinase substrates exhibited a large shift in density parallel with markers for the plasma membrane, whereas markers for the other organelles exhibited smaller shifts in density. In conjunction with the findings of the accompanying paper (Casnellie, J. E., Ives, H. E., Jamieson, J. D., and Greengard, P. (1980) J. BioL Chem 255, 3770-3776), these findings suggest that the substrates for cGMP-dependent protein kinase are integral proteins of the plasma membrane, while the enzyme itself appears to exist in both a soluble form and as a peripheral plasma membrane protein.

Cyclic GMP-dependent protein kinase and endogenous sub-

* This study was supported by United States Public Health Service Grants GM-21714 (to J. D. J.) and DA-01627, MH-17387, and NS- 08440 (to P. G.) and a grant from the McKnight Foundation (to P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Supported by a scholarship from the North American Reassur- ance Co., New York, NY through the Insurance Medical Scientist Schola-rship Fund, Springfield, MA and by the United States Public Health Service Medical Scientist Training Program Grant GM-02044. Present address, Department of Internal Medicine, Columbia Uni- versity College of Physicians and Surgeons, New York, NY 10032.

Washington, Seattle, WA 98105. 0 Present address, Department of Pharmacology, University of

1 To whom correspondence should be addressed.

strates for this enzyme have been demonstrated in the total particulate fraction from rabbit aortic medial smooth muscle (1). Since little is known about the function of cGMP or cGMP-dependent protein kinase in smooth muscle, it is im- portant to establish more precisely the subcellular location of both the enzyme and its substrates.

A number of fractionation schemes have been developed for intact vascular smooth muscle (2-5) and for isolated vascular smooth muscle cells (6, 7), but these were either unsuitable for the present study or the distribution of organelles was only partially characterized. We have, therefore, devised a new homogenization and fractionation scheme for the rabbit aortic media which results in partial separation of the major subcellular components without significant losses, based on the recovery of enzymatic markers. Using this new fractionation procedure, we have studied the subcellular localization of the particulate cGMP-dependent protein kinase and its substrates.

EXPERIMENTAL PROCEDURES

Materials-5‘-AMP, thymidine 5“monophospho-p-nitrophenyl ester, UDP-galactose, cytochrome c, ascorbic acid, p-nitrophenyl phosphate, p-nitrophenol, ouabain, antimycin, disodium succinate, Trizma/HCI, digitonin, and Triton X-100 were purchased from Sigma Chemical Corp. [y-32P]ATP was purchased either from ICN or Amer- sham and diluted to a specific activity of 3 to 10 X lo7 cpm/nmol. 8- NS-[32P]~IMP was prepared as previously described (1). Glutaralde- hyde, osmium tetroxide, and Epon were purchased from Electron Microscopy Sciences. /3-Mercaptoethanol was purchased from Bio- Rad Laboratories and stored at 4OC for no longer than 1 month. All other materials were obtained as in Ref. 1.

The standard buffer consisted of 8.5% (w/v) sucrose, 10 mM Hepes’ (pH 7.4), 0.2 mM MgC12, and 10 mM P-mercaptoethanol. It was kept at 4°C and used for only 24 h. Scintillation fluid (4 liters) consisted of 3 liters of xylene, 1 liter of Triton X-114, 12 g of 2,5-diphenyloxazole (PPO), 0.8 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP). Aqua- sol from New England Nuclear was used for liquid scintillation counting of dissolved gel slices.

Homogenization-All phases of the homogenization and fractionation procedure were carried out at 4°C. Slices (0.5 mmZ) of aortic medial smooth muscle tissue from four rabbits were prepared as described (l), suspended in 3 ml of the standard buffer, and homog- enized in a Brendler-type tissue grinder, Size A (Arthur H. Thomas), With a wall clearance of 0.10 to 0.15 mm. The pestle was driven at a loaded rpm of 2200 by a %-horsepower Bodine Fractional Horsepower Motor type NSE 34 (Bodine Electric Go.) with a Powerstat Variable Autotransformer type VS3PN116B (Superior Electric Co.) set a t 80 V AC. The suspension of aortic medial slices was subjected to one up- and-down stroke which lasted approximately 15 s. The resulting slurry was then passed through 10 pm Nytex gauze (Tekto, Inc.). The homogenizer was washed with 1 ml of standard buffer; this wash was then used to resuspend the residue on the filter for refiltration. The ffitrates were combined and termed the homogenate, while the resi-

I The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid; 8-N3-cIMP, 8-azidoinosine 3’:5”monophosphate; 8-N3-cAMP, 8-azidoadenosine 3’:5”monophosphate.

3777

3778 Subcellular Location of cGMP-dependent Protein Kinase

due, which consisted of large pieces of acellular connective tissue as visualized by phase and Nomarski differential interference contrast microscopy, was discarded. Strict adherence to this procedure was necessary for reproducibility of the fractionation results.

Sucrose Density Gradient Fractionation-Three milliliters of the homogenate was loaded onto a 9-ml linear 8.5 to 60% (w/v) sucrose gradient containing 10 mM Hepes (pH 7.4), 10 mM P-mercaptoethanol, and 0.2 mM MgC12 in a cellulose nitrate centrifuge tube (% X 3% inches) and centrifuged for 3 h at 40,000 rpm in the SW 41 rotor of a Beckman (Spinco Division, Beckman Instruments) L5-65 ultracentri- fuge with the brake off. Twelve fractions of 1 ml each were collected with a Buchler Auto Densi-Flow (Buchler Instruments Division of Nuclear-Chicago) and an LKB peristaltic pump. The density of each fraction was calculated from the refractive index determined with a Bausch & Lomb Abbe refractometer. The pellet from the sucrose gradient fractionation was resuspended in 1 ml of the standard buffer and termed the pellet fraction.

In those gradient fractionation experiments in which digitonin was used, the digitonin (approximately 80% pure) was dissolved at 2.2 mg/ ml in the standard buffer using gentle heating until the solution became clear. This material was then added dropwise to homogenates to achieve a final concentration of 280 pg of digitonin/mg of homogenate protein (see “Results”). Fractionations were then carried out as above.

Enzyme and Other Chemical Assays-Endogenous protein phosphorylation was carried out using 25 to 150 pg of protein (fractions or homogenate) and quantitation of ”P incorporation into specific bands was determined as in Ref. 1. In the experiments of Table I, “*P incorporation into protein GI in the presence of cGMP was expressed as picomoles/mg of protein. The data have not been corrected for phosphorylation in the absence of cGMP.

Photoaffinity labeling with 8-N&*P]cIMP was performed using 25 to 150 pg of protein and incorporation of radioactive label into specific bands was measured as in Ref. 1. In some experiments, quantitation of incorporated label was carried out by densitometry using a Canalco model G I1 microdensitometer as previously described (8).

Galactosyltransferase was measured as previously described (9) using incorporation of galactose from UDP-galactose (galactose-l- ’H(N)) into asialo-agalactofetuin. Protein (5 to 25 pg) was incubated in a final volume of 100 pl containing 30 mM sodium cacodylate buffer (pH 7.4). 30 mM MnCL, 30 mM P-mercaptoethanol, 0.2% Triton x- 100, 2 mM ATP, 200 pg of asialo-agalactofetuin, and 4 p~ UDP- galactose (gala~tose-l-~H(N)) (40 mCi/mmol). Incubation was carried out for 30 rnin at 37OC and stopped by the addition of 1.0 ml of 1% phosphotungstic acid in 0.5 N HCI at 4°C. After 1 h, tubes were centrifuged at 2000 rpm for 10 min in a Damon/IEC PR-6000 centrifuge. Supernatants were discarded and pellets were washed twice with 1% phosphotungstic acid in 0.5 N HCI and once with absolute ethanol (-10°C). Each pellet was then dissolved in 0.5 ml of 2% sodium dodecyl sulfate, and the resulting solution was added to 10 ml of standard scintillation fluid and counted for 10 min in a Beckman LS-250 liquid scintillation spectrometer (Beckman Instruments).

Although we have not measured the consumption of UDP-galactose under these conditions, less than 5% of the galactose initially present in UDP-galactose was incorporated into asialo-agalactofetui, and the reaction was linear for 30 min with 5 to 50 pg of protein. Since less than saturating concentrations of radioactive substrate were used for this assay to increase its sensitivity and decrease its cost, activity is reported as cpm incorporated/min/mg of protein rather than as picomoles of substrate incorporated.

5”AMPase was assayed by a modification of previously described methods (10). Cleavage of P, from 5’-AMP was measured by incubation of 5 to 25 pg of protein in a final volume of 1.0 ml containing 100 mM Trizma/HCl (pH 8.0), 10 mM MgC12, and 5 m~ 5’-AMP. The reaction mixture was incubated for 30 min at 37°C and the reaction was stopped by the addition of 0.2 ml of 30% trichloroacetic acid at 4°C. After 30-min incubation on ice, the samples were centrifuged at 2000 rpm for 10 min as for the galactosyltransferase assay. The supernatant, (0.4 m l ) was removed and assayed for inorganic phosphate by a modification (11) of the Fiske and Subbarow (12) assay. A solution (0.1 m l ) containing 1.4% ascorbic acid and 0.4% ammonium molybdate in 1 N sulfuric acid was added to each sample. After a 20- min incubation at 45OC, the absorbance of each sample was read at 750 nm in a Beckman model 25 spectrophotometer. Results were expressed as nanomoles of phosphate released/min/mg of cell protein.

Alkaline phosphodiesterase was assayed by a modification of previously described methods (13). The cleavage of thymidine 5’-monophospho-p-nitrophenyl ester was measured by the appearance of p-

nitrophenol. Protein (5 to 25 pg) was incubated in a final volume of 0.5 ml containing 100 mM glycine/NaOH buffer (pH 10.0), 0.1% Triton X-100, 2 mM zinc acetate, and 1.5 mM thymidine 5”monophospho-p- nitrophenyl ester. After a 15-min incubation at 37”C, the reaction was stopped by the addition of 1.0 ml of 0.1 N NaOH. Absorbance at 410 nm was read on a Beckman model 25 spectrophotometer. Results were expressed as nanomoles of p-nitrophenol released/min/mg of protein.

Acid phosphatase was measured by the release of p-nitrophenol from p-nitrophenyl phosphate. Protein, (25 to 125 pg) was incubated in a final volume of 0.5 ml containing 50 mM sodium acetate buffer (pH 5.0), 1 mM p-nitrophenyl phosphate, 0.1% Triton X-100. After a 30-min incubation at 37”C, the reaction was stopped by 1.0 ml of 0.1 N NaOH and the absorbance at 410 nm was read as above. Results were expressed as nanomoles of p-nitrophenol released/min/mg of cell protein.

Succinate cytochrome c reductase was measured by modification of previously described methods (14). The reduction of cytochrome c was followed by measuring its increase in absorbance at 550 nm. Protein (50 to 25 pg) was incubated in a final volume of 1.0 ml containing 40 mM sodium phosphate buffer (pH 7.4), 20 m~ sodium succinate, 1 m~ potassium cyanide, and 1.5 mg of cytochrome c in the presence or absence of 0.13 pg/ml of antimycin. The reaction was started by the addition of the protein and the change in absorbance at 550 nm was followed for 5 min in a Beckman Acta I11 recording spectrophotometer. Antimycin-sensitive cytochrome c reduction was expressed as nanomoles of cytochrome c reduced/min/mg of protein using a millimolar extinction coefficient of E?z - E:’ = 18.5.

NADPH cytochrome P-450 reductase was measured by reduction of cytochrome c and is therefore referred to as NADPH-cytochrome c reductase. Assay conditions were as described for succinate cytochrome c reductase except that succinate and antimycin were omitted and 3 mM NADPH was included.

RNA was assayed by previously described methods (15). Samples were precipitated with 0.5 M perchloric acid and the pellets were hydrolyzed for 60 min at 37°C in 0.6 N KOH. The absorbance at 260 nm of the supernatant of a second precipitation with 0.5 M perchloric acid was read in a Beckman model 25 spectrophotometer and the RNA concentration was determined using an E?p of 312. Controls containing bovine serum albumin and DNA indicated no interference from these substances under the conditions outlined.

DNA was assayed by the method of Burton (16) after hydrolysis for 20 min at 80°C in 0.5 M perchloric acid using calf thymus DNA as standard.

Protein was assayed by a modification of the Bio-Rad protein assay (17). Samples containing 5 to 20 pg of protein were diluted to 20 pI and added to 0.8 ml of the Bio-Rad reagent (Bio-Rad Laboratories) which had been diluted with 4 parts of H20. The assay was otherwise as described (17). Bovine y-globulin was used as standard.

Electron Microscopic Procedures-Fractions from the sucrose gradient were centrifuged for 10 min at 50,000 rpm in a Beckman SW 50.1 rotor. The pellets were fried in 1% osmium tetroxide in 0.1 M sodium cacodylate (pH 7.4) for 30 min at 4”C, washed at 4°C with isotonic saline (0.9% NaCl solution), and stained in block with 0.5% magnesium uranyl acetate in isotonic saline for 2 h at 25%. The pellets were then dehydrated in ethanol and propylene oxide and embedded in Epon (18). Thin sections were doubly stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop 101 at an operating voltage of 80 kV. Cell pellets were oriented during embedding so that they could be examined from top to bottom.

RESULTS

Homogenization and Fractionation Procedure-Prelimi- nary studies indicated that cGMP-dependent protein phosphorylation in particulate fractions was rather labile in that the phosphorylation in homogenates was reduced by approximately 50% after storage for 8 h at 4°C. Therefore, we devised a homogenization and fractionation procedure in which the time involved and the number of manipulations were kept to a minimum (see “Experimental Procedures”). Fresh 10 mM P-mercaptoethanol was found necessary to maintain enzyme activity and 280 pg of digitonin/mg of protein appeared to prevent losses during the fractionation procedure (see below). Minor deviations from the procedure described under “Ex- perimental Procedures,” such as centrifugation and resuspen-

Subcellular Location of cGMP-dependent Protein Kinase 3779

Homogenate 1.03 1.03 1.04 1.05 1.08 1 . 1 I 1.12

1.14 O G

O G O G O G O G O G O G O G - " . "

-60

" G I

JG2 "43

1.15 1.16 1.18 1.20 1.21 1.22 1.23 Pellet O G O G O G O G 0 G 0 G O G O G

FIG. 1. Endogenous cGMP-dependent protein phosphorylation in fractions from sucrose density gradients. Fractions were prepared by the standard procedure and endogenous phosphorylation, gel electrophoresis, and autoradiography were carried out on 50 pl from each sample in the absence (0) or presence ( G ) of 5 X 10 ' M cGMP. Note the co-sedimentation of proteins Go to G:,.

sion of the individual fractions, caused considerable loss of activity. This homogenization procedure for aortic media smooth muscle was compared with a tissue homogenization procedure which was more vigorous and with a procedure which involved Dounce homogenization of isolated cells prepared as described previously (1). The gentle procedure for homogenization of the tissue produced narrower peaks of marker enzyme activity in density gradient experiments than either of the other procedures and thus appears to be advan- tageous for cell fractionation studies.

Distribution of Substrates for cGMP-dependent Protein Kinase in Sucrose Density Gradients-When fractions from the sucrose density gradient fractionation procedure were examined for endogenous cGMP-dependent protein phosphorylation, substrate proteins Go to Gi1 (described in Ref. 1) co- sedimented in a peak with density range from 1.08 to 1.15 (Fig. 1). So that these results could be quantitated, conditions were established under which the total incorporation of "'P into protein GI (the most heavily labeled substrate) was linear (Fig. 2) with the amount of protein (derived from the total homogenate or its fractions). The phosphorylation of protein GI was exceedingly rapid even a t 4°C and it was therefore

impossible to measure the rate of this reaction. Total "'P incorporated into protein GI at this temperature was constant after 10 s and for ATP concentrations between 5 and 100 p ~ . Since degradation of ATP by ATPases in 20 s was as much as 85% a t 30°C but only 25% a t 4"C, all reactions were carried out at 4°C and with 10 PM ATP to assure that protein GI was maximally phosphorylated. Since it was not determined whether all phosphate acceptor sites on protein GI were available (Le. some sites may have been phosphorylated in uiuo), this saturated level does not necessarily represent the total number of protein G I molecules.

Phosphorylation of substrate protein GI in a typical fractionation experiment is plotted in Fig. 4 as a function of density in the gradient; the median density of particles containing this phosphorylated substrates was 1.10 g/ml.

Any protein GI that may have been present in the pellet was largely obscured in most experiments ( e g . Fig. 1) by a highly radioactive band that migrated in the protein GI region of the gel. The phosphorylation of this protein(s) showed little or no cGMP dependency. Nevertheless, in all experiments, protein GI in the pellet was defined based on electrophoretic mobility and was included in the quantitation (Fig. 4) for

37ao Subcellular Location of cGMP-dependent Protein Kinase

completeness even though it probably consisted, at least in part, of protein(s) different from protein GI.

Despite the known linearity of the phosphorylation reaction with protein concentration in the membrane fractions (Fig. 2), the recovery of '"P incorporated into protein GI in the fractions was reproducibly only 20 to 40% of that in the starting homogenate when fractionation was carried out in the absence of digitonin. However, recovery was reproducibly greater than 80% when homogenates were treated with 280 pg of digitonin/ mg of protein prior to fractionation (see below). This was almost entirely due to increased incorporation of snP in the fractions rather than to decreased incorporation in the homogenate (see Table I), and was peculiar to the phosphorylation reaction since other enzyme markers were not similarly affected (see legends to Figs. 4 and 5). Various potential causes

V I 1 1 1 25 so 75 100

PROTEIN ( u o ) FIG. 2. 32P incorporation into protein GI as a function of

protein concentration. Fractionation of aortic medial smooth muscle was carried out by the standard procedure and fractions of density 1.09 to 1.13 were pooled. Endogenous phosphorylation was carried out by the standard procedure using ATP with a specific activity of 5 X 10' cpm/nmol and various amounts of protein as indicated. 'lP incorporation was determined by liquid scintillation spectrometry of the gel slice corresponding to protein GI as described under "Experi- mental Procedures."

Hornog

Origin

? 1 x

c 0

c 100-

.- 0 7 5 -

47- - :: 50-

74- L O 2 -

54- 0 I

40-

I .03 1.04 1.06 1.07

for the loss of activity in the absence of digitonin were inves- tigated.

1) The addition of from 100 to 10oO pg of digitonin/mg of protein to fractions prepared in the absence of digitonin did not alter the yield of activity in these fractions, suggesting that digitonin exerts its action during the fractionation procedure.

2) The loss of activity was not observed if the homogenates in 8.5% sucrose were simply centrifuged for 1 h a t 160,OOO X g as in Ref. 1, nor was the activity lost if homogenates were diluted with 3 volumes of standard buffer and then incubated for 4 h at 4°C in a cellulose nitrate tube. This implies that the removal of soluble factors from the particulate substrates is not responsible for the loss of activity and suggests that some aspect of the sucrose gradient procedure is causing the low yield. Thus, the loss of activity could result from the prolonged exposure to the pressure generated during centrifugation, interaction of the particles with the walls of the tube during sedimentation, or the manipulations involved in harvesting the fractions.

3) The yield was not increased by addition of 0.5% Triton X-100 to the fractions, suggesting that limited accessibility of ATP or Mg2' during the reaction was not a problem.

4) The addition of excess purified aortic cCMP-dependent protein kinase (19) or whole cytosol (prepared as in Ref. l), either in the presence or absence of 0.5% Triton X-100, was also without effect, again suggesting that removal of cytosolic factors during centrifugation did not play a role in the loss of activity.

5) Mixing of fractions enriched in protein GI from fractionations performed in the presence and absence of digitonin demonstrated only simple additivity in the amount of "P incorporated into this protein, indicating that a loosely bound inhibitory or stimulatory factor was not present in either set of fractions.

6) When whole homogenates were reconstituted by remix- ing the entire set of fractions (including the pellet) obtained either with or without digitonin, the total recovery was un- changed from that obtained by examining the fractions sepa- rately. This further argues against the separation of either inhibitory or stimulatory factors during centrifugation as an

DENSITY 1.09 1.1 I 1.13 1.15 1.17 1.20 1.22 Pel

FIG. 3. Autoradiography showing labeling pattern with 8-N3-[32PJcIMP in fractions from sucrose density gradients. Fractions of the indicated densities were prepared by the standard procedure and photoaffinity labeling was carried out as described under "Experimental Procedures" using S-N,,-[:"P]cIMP with a specific activity of 2 X 10' cprn/nrnol.


EOUlLlBRlUM DENSITY DENSITY

FIG. 4 (left). Frequency-density distribution histograms of cGMP-dependent phosphorylation of protein GI, photoaffinity labeling of the 47,000- and 74,000-dalton proteins with B-N3- [32P]cIMP, enzymatic markers, RNA, DNA, and protein. Frac- tionation of aortic medial smooth muscle was carried out by the standard procedure and all assays were performed as described under "Experimental Procedures." Thin vertical line at the bottom of each histogram represents the median density of 32P incorporation into protein GI (based on activity present between densities of 1.07 to 1.21). Recovery of activity in the gradient (including the load and pellet) was: 3zP incorporation into protein GI, 25% 8-N3-[32P]cIMP labeling of 74,000-dalton band, 12276, and of 47,000-dalton band, 118%; alkaline phosphodiesterase, 99%; 5'-AMPase, 85% galactosyltransferase, 85% NADPH-cytochrome c (cyt. c) reductase, 68%; acid phosphatase, 90%; succinate cytochrome c (cyt. c) reductase, 75%; RNA, 107%; protein, 95%; DNA, 90%. Results plotted are the average of duplicate determinations in a single experiment representative of four similar experiments.

FIG. 5 (right). Frequency-density distribution histograms of P incorporation into protein GI and enzymatic markers in

fractionations performed with and without digitonin. Homog- enates were treated with digitonin as described in the text and fractionation was otherwise by the standard procedure. Stippled histograms, data reproduced from Fig. 4; histograms with thick line, distribution after digitonin treatment; arrows, median densities of components from digitonin-treated (black arrows) or untreated (white arrows) homogenates. Recovery of enzyme activity in the digitonin-treated samples was: J2P incorporation into protein GI, 82%; alkaline phosphodiesterase, 86%; galactosyltransferase, 81%; NADPH-cytochrome c (cyt.c) reductase, 69%. Results plotted are the average of duplicate determinations from a single experiment. Similar results were obtained in several additional experiments (for numbers of experiments, see Table 11).

32

explanation for the poor recovery in the absence of digitonin. Thus, the mechanism by which digitonin appeared to protect against loss, during density gradient centrifugation, of the ability to incorporate 32P into protein GI remains unexplained.

Distribution of cGMP-dependent Protein Kinase in Su- crose Density Gradients-The phosphorylation of endoge-

nous substrates does not provide a quantitative measure of cGMP-dependent protein kinase activity in the gradient fractions because such phosphorylation is too rapid (see above) to determine its rate and because the substrates are not evenly distributed in the gradient. In addition, in the present study we have found histone 11-A, a convenient substrate for the soluble cGMP-dependent protein kinase (19, 20), to be too insensitive as a substrate for assay of the particulate enzyme. Therefore, the distribution of the kinase was studied directly by labeling its cyclic nucleotide binding site with the photoaffinity ligand 8-N3-[32P]cIMP. Since this agent also labels the regulatory subunit of the type I and type I1 CAMP-dependent protein kinases (19), the distributions of these two enzymes were also determined.

The pattern of proteins labeled with 8-N3-[32P]cIMP in the homogenate and gradient fractions is shown in Fig. 3. The 74,000-dalton protein has previously been shown to be the subunit of the cGMP-dependent protein kinase and the 47,000- and 54,000-dalton proteins have been identified as the regulatory subunits of the type I and type I1 CAMP-dependent protein kinases, respectively, in the cytosol fraction of vascular smooth muscle (19). The 40,000-dalton protein appears to be a breakdown product of the type I1 regulatory subunit.' In- corporation of radioactivity into the band immediately below the 47,000-dalton protein was not inhibited by either CAMP or cGMP and therefore represents nonspecific labeling.

The distribution of these three labeled bands in the gradient was quite different. The 74,000-dalton cGMP-dependent protein kinase subunit was present primarily at the top of the gradient (with soluble proteins); a second peak, of median density 1.12, was found in approximately the same region of the gradient as that containing the endogenous substrate proteins. The 47,000- and 54,000-dalton bands were almost entirely at the top of the gradient and rapidly disappeared toward the denser fractions. To show that the association of the 74,000-dalton protein with cell particles of density 1.12 was not due to artifactual adsorption of the soluble enzyme (to either the external or cytosolic surface of the plasma membrane (see below) or to any other membranous particles), prelabeled soluble enzyme previously purified from rabbit aortic media (19) was added to aortic medial slices prior to homogenization. This labeled material behaved in the same manner as the 47,000- and 54,000-dalton proteins, i.e. there was no apparent adsorption to any cell particles.

Since 32P (in the form of 8-N3-["'P]cIMP) incorporation into the membrane fractions was too low for accurate scintillation counting, these results were quantitated by densitometric scans of the gel autoradiograms (Fig. 4). In each of three experiments, this quantitative measurement indicated that the median density of the second peak of the 74,000-dalton protein was higher by 0.02 than the peak containing the substrates. Further analysis and discussion of this difference is presented below.

Distribution of Enzymatic Markers, RNA, and DNA in Sucrose Density Gradients-Since the densities of organelles from smooth muscle have not been definitively established and our fractions were prepared differently from previously published procedures, studies were carried out to charactelize our fractions both by enzyme markers and by morphology. Density distributions of the markers are shown in Fig. 4, their median densities are tabulated in Table 11, and their specific activities are given in Table I. Several markers had a grossly different distribution from that of the membrane-associated cGMP-dependent protein kinase and its substrates. DNA (Fig. 4) and morphologically identifiable nuclei (Fig. 6) were found

* U. Walter, personal communication.


TABLE I Specific activity of enzymatic markers in aortic medial smooth

muscle Specific activities in homogenate and peak fractions (density of

peak fractions given in brackets) were calculated from data accumu- lated from six fractionation experiments using the standard procedure. Numbers represent mean f S I > . for the number of experiments indicated in parentheses. Units used for each enzyme are described under "Exoerimental Procedures."

Enzyme Specific activity

Homoeenate Peak

: V incorporation into protein G I

- Digitonin + Digitonin

Galactosyltransferase 5"AMI'ase Alkaline phosphodiesterase NADPH-cytochrome c re-

ductase Acid phosphatase Succinate cytochrome c re-

ductase

0.5 f O . l (3) 1.0 f O . l 0.4 f 0.1 (3) 3.5 f 0.5 62 k 22 (3) 371 k 66

1 2 5 f 14 (3) 652 f 100 95 f 14 (4) 753 f 2 2 1 3.4 f 0.9 (3) 10.9 f 1.7

[1.10]

[1.11] [1.12] [1.12]

[1.14]

11.141

7.9 f 2.0 (3) 32.4 f 8.7 [1.15] 5.1 k0 .7 (3) 33.8 k 11.0 11.171

TABLE I1 Median densities of enzymatic markers in the presence and

absence of diEitonin Increase in median density

+ dieitonin Y

activity Median density - digitonin Experiment

1 2 3 ;x2 P incorporation

into protein GI Alkaline phosphodies-

5"AMPase terase

8-N:t-[:"'P]cIMP label in 74,000-dalton band

Galactosyltransferase NADPH-cytochrome

c reductase Acid phosphatase Succinate cyto-

chrome c reductase

1.100 f 0.004 (3) 0.042 0.032 0.046

1.125 f 0.010 (4) 0.041 0.029 0.053

1.123 f 0.009 (3) 0.038 1.128 f 0.002 (3) ND" ND ND

1.117 k 0.005 (3) 0.028 0.013 1.140 f 0.012 (2) 0.003 -0.005

1.150 f 0.014 (3) 0.030 1.165 f 0.005 (4) 0.005

" ND, not determined (see text).

1.06 - 1.13 D E N S I T Y 1.14-1.17 0 5 p m

FIG. 6. Electron micrographs of pellets from pooled gradient fractions. Samples were prepared and processed for electron microscopy as described in the text. a, region near the top of the pellet from pooled fractions of density 1.06 to 1.13. Note smooth vesicles of various sizes and the free ends (fe) of membrane sheets. X 23,000.6, region near the bottom of the same pellet as depicted in a . Note contamination of this membrane fraction with lysosomes (ly) and multivesicular bodies (mu). X 23,000. c, region near the top of the pellet from pooled fractions of density 1.14 to 1.17. Smooth membranes, filamentous material (fi), rough microsomes (rm), and mitochondria can be seen. X 23,000. d , region near the bottom of the

same pellet as depicted in c. Numerous mitochondria, filamentous material, and rough microsomes are present. X 23,000. e, region near the top of the pellet from pooled fractions of density 1.18 to 1.22. Mitochondria, filamentous material, and rough microsomes predominate. X 23,000. f , region near the bottom of the same pellet as depicted in e. Rough microsomes predominate, with some contamination by mitochondria and other cellular elements. X 23,000. g, micrograph representative of the bottom portion of the gradient pellet, depicting an essentially pure preparation of nuclei. Upper 10 to 20% of this pellet (not shown) contained aggregated material similar to that seen in e and f. X 5,800.

Subcel lu la r Location of cGMP-dependent Protein Kinase 3783

only in the pellet. Structures containing RNA, a marker for rough microsomes and free ribosomes, and succinate cytochrome c reductase, an inner mitochondrial membrane marker, were considerably denser than structures containing the kinase and substrates. Acid phosphatase, a lysosomal marker, showed a bimodal distribution (excluding the pellet), conceivably due to the breakage of lysosomes during homogenization. However, the median density for presumably intact lysosomes (1.15) was significantly higher than that for structures containing cGMP-dependent protein kinase.

The second peak of the 74,000-dalton protein corresponded most closely in position with the peaks of 5”AMPase and alkaline phosphodiesterase, the markers for plasma membrane. However, galactosyltransferase, a Golgi marker, and NADPH-cytochrome c reductase, a marker for endoplasmic (sarcoplasmic) reticulum (see “Discussion”), had distributions that were very close to those of the plasma membrane markers. Because of the lack of resolution of these components and because the kinase substrates had a lighter distribution than the enzyme itself, further gradient analyses were carried out with homogenates pretreated with digitonin.

Effect of Digitonin Treatment on the Distribution of cGMP-dependent Protein Kinase, Substrates, a n d Enzy- matic Markers in Sucrose Density Gradients-Digitonin has been shown to increase the density of certain cell particles in a number of tissues, including liver (13) and cultured aortic endothelial and smooth muscle cells (7). This effect appears to be due to the cholesterol binding properties of digitonin (13) and the degree of shift obtained is presumed to reflect the cholesterol content of the cell particles. The use of 280 pg

~ ~. ~ - ” . ~.

of digitonin (0.18 pmol, assuming 80% purity)/mg of homogenate protein (see “Experimental Procedures”) was designed to achieve a 1:l mol ratio of digitonin to cholesterol based on the finding of 0.18 pmol of cholesterol/mg of protein in aorta (21). Digitonin treatment shifted the distribution of substrates for cGMP-dependent protein kinase and plasma membrane markers considerably, and by approximately the same amount (0.04 g/ml) (Table 11; Fig. 5). Thus, the median density of fractions containing the substrates was lighter by 0.02 g/ml than the median density of fractions containing plasma membrane markers in the presence, as in the absence (see above), of digitonin. All other markers examined exhibited smaller shifts (Table 11). Since the particles containing the substrates thus appeared to have the same cholesterol content as that of plasma membrane and the other organelles appeared to have lower cholesterol contents, we feel that the substrates for cGMP-dependent protein kinase are in the plasma membrane, although apparently in a light plasma membrane subfraction. When taken in conjunction with the findings presented in the accompanying paper (l), the substrates would appear to be integral plasma membrane proteins. Alternative interpreta- tions of these findings will be discussed below.

Studies with 8-Ns-[”’P]cIMP labeling revealed that treatment with digitonin also shifted the second peak of cGMP- dependent protein kinase to fractions of higher density in the gradient (data not shown) as compared to fractions without digitonin treatment. However, in addition, digitonin treatment also broadened the second peak of this activity and conse- quently its median density could not be accurately determined.

”

1.18- 1.22 0.5 pm Pel le t ~

2.0 pm


Morphology of Fractions from Sucrose Density Gra- dients-The morphology of several groups of pooled fractions was studied by electron microscopy. Fractions of density 1.06 to 1.13, 1.14 to 1.17, and 1.18 to 1.22 were combined, pelleted, and processed for electron microscopy along with the gradient pellet. Representative fields from each pellet are shown in Fig. 6. Fractions of density 1.06 to 1.13 (Fig. 6, a and 6 ) which contained the bulk of the cGMP-dependent protein kinase, substrates, and plasma membrane markers consisted primarily of large sheets of smooth membranes and smooth vesicles of various sizes. Vesicles were never fused with sheets as might be expected based on the presence of numerous caveolae on the plasma membrane of intact tissue (22). Small amounts of contamination by lysosomes, mitochondria, and rough endoplasmic reticulum were also observed in these fractions. Frac- tions of density 1.14 to 1.17 (Fig. 6, c and d ) contained smooth membranes, but there were considerably greater numbers of mitochondria (in varying states of preservation), lysosomes, and filamentous material than in the lighter fraction. Frac- tions of density 1.18 to 1.22 (Fig. 6, e and f ) contained large numbers of mitochondria, lysosomes, and vesicles of rough endoplasmic reticulum along with some filamentous material. The pellet contained two discrete regions with a sharp de- marcation between them. The upper portion contained a distribution of particles similar to that seen in Fig. 6h except that they were more aggregated (not shown). The lower portion consisted entirely of nuclei (Fig. 6g). The morphological appearance of the fractions thus was consistent with the distribution of marker enzymes, especially in that the fractions lighter than 1.14 g/ml consisted almost entirely of various types of smooth membranes.

DISCUSSION

Although soluble cGMP-dependent protein kinase from aorta appeared to be relatively stable (19, 23), the phosphorylation reaction in particulate fractions had a rather short half-life at 4°C and was sensitive to some aspects of the procedure involved in fractionating the homogenates in a sucrose gradient. The loss of activity which took place during sucrose gradient centrifugation was prevented by the cholesterol binding agent, digitonin.

Since we were unable to reconstitute lost activity in the fractions by the addition of detergents, purified soluble enzyme, or whole cytosol (160,000 X g supernatant), or by recombining the entire set of gradient fractions, it would appear that separation of components necessary for the reaction during gradient centrifugation cannot explain the inabil- ity to obtain full recovery of activity in the absence of digitonin. Moreover, the fact that virtually full recovery was obtained in fractionations performed with digitonin argues that all cellular factors necessary for the reaction are present in the gradient fractions enriched in cGMP-dependent protein kinase activity. It is conceivable that the substrates or enzyme may be denatured during prolonged centrifugation or incubation in dilute solutions. The mechanism by which digitonin protects against this inactivation has no simple explanation.

Because of the lability of the phosphorylation reaction, we have devised a new fractionation procedure involving very mild homogenization and only one centrifugation step. As a by-product of this study, we have found that mild homogenization of the tissue results in higher specific activities and better separation of various enzyme markers than more vigorous homogenization without significant decrease in the total yield of enzyme activities. This is in contradiction to the findings of other workers (2, 6) who have suggested that extremely vigorous homogenization is necessary to release

subcellular organelles from vascular tissue and that single vascular smooth muscle cells are easier to homogenize. We have found that it is difficult to homogenize single cells adequately and that, at best, they provide only small amounts of material. Thus, this new homogenization and fractionation scheme should prove useful for future subcellular distribution studies in vascular smooth muscle.

Our claim that cGMP-dependent protein phosphorylation takes place in plasma membrane rather than sarcoplasmic reticulum is based on the assumption that NADPH-cytochrome c reductase is a marker for sarcoplasmic reticulum, since we have had difficulty demonstrating significant Ca2+- dependent ATPase activity in our fractions as have others using different fractionation schemes (2,24). This assumption may be criticized since there is little evidence that the enzyme is present in sarcoplasmic reticulum. However, NADPH-cytochrome c reductase has been well established as a marker for rough and smooth endoplasmic reticulum from liver (25), although recently small amounts of the total enzyme activity have been convincingly shown to be associated with Golgi membranes (9,26). Magargal et al. (7) found that this enzyme is present in a light membrane fraction from cultured smooth muscle ( p = 1.14) and that its density does not shift in the presence of digitonin as has been confirmed by us using homogenates from intact aorta. These findings suggest that NADPH-cytochrome c reductase is present on smooth membranes other than Golgi or plasma membrane (both of which shift in density after digitonin treatment). Such smooth membranes have all been traditionally classified as sarcoplasmic reticulum morphologically (22), and all appear to accumulate strontium (27), although there is no evidence that they are biochemically homogeneous or that they all sequester Ca2'. We have assumed that NADPH-cytochrome c reductase is present in the same membrane-bound compartments which sequester Ca"' on the basis of the above arguments. If our assumptions are valid, digitonin treatment of the homogenate followed by fractionation provides a potential first step in the purification of sarcoplasmic reticulum from smooth muscle cells, since under these conditions it is slightly lighter than plasma membranes or Golgi membranes (see Fig. 5), the major potential contaminants of a preparation of sarcoplasmic reticulum.

As an additional by-product of this study, we have also been able to determine the subcellular distribution of the CAMP- dependent protein kinases. As can be seen in Fig. 3, both type I and type I1 regulatory subunits appear to be entirely soluble.

The distribution of the cGMP-dependent protein kinase was very different from that of the CAMP-dependent enzymes. Approximately 25% of the cGMP-dependent enzyme (based on binding of 8-N3-[32P]cIMP) is particulate. The particulate enzyme cannot be washed off by either low salt or isotonic (0.125 M KC1) salt solutions (1). In this paper, we have determined that the particulate enzyme is most likely localized to the plasma membrane, based on its sedimentation in sucrose density gradients; however, this enzyme may also be present in small amounts on other membranes since there was a broadening of its distribution after digitonin treatment (data not shown).

In contradistinction to our findings on the distribution of cGMP-dependent protein kinase, the substrates for this enzyme have a somewhat anomalous distribution. Our studies indicate that the substrates are present in membranes which have a lighter median density than any of the marker enzymes we have examined, but that they respond to digitonin similarly to plasma membrane (i.e. they most likely are in membranes that have the same cholesterol content as plasma membrane).


There are at least three possible explanations for these obser- vations. 1) Phosphorylation takes place on a subfraction of the plasma membrane which is either slightly less dense than the bulk or fails to come to equilibrium in the short (4 h) centrifugation necessary to preserve activity of the phosphorylation reaction, but the kinase is bound to the bulk of plasma membrane as well as the subfraction. 2) Phosphorylation takes place on a previously unidentified membrane which represents only a small fraction of the total and which contains only small quantities of the marker enzymes we have examined, but the kinase is more widely distributed. 3) The phosphorylation reaction takes place in several organelles and its distribution therefore does not parallel any single one of them. The second explanation is difficult to disprove since it states that there would be no independent means for detection of the hypothetical membrane; the third seems somewhat un- likely in view of the rather narrow distribution of the substrate proteins (Fig. 4) and their extremely low median density (1.10); however, there is rationale for accepting the first explanation.

Smooth muscle plasma membrane contains many caveolae which are believed to be a specialization of the plasma membrane since they are always in communication with the extra- cellular space (22). Because of the narrow necks by which many of these caveolae are attached to the plasma membrane (22), it is conceivable that they could be easily pinched off during homogenization. Such vesicles (formed from pinched off caveolae) may then migrate differently from the bulk of plasma membrane because of their shape, the density of their content, or the amount of protein adhering to them. Subfrac- tionations of the plasma membrane based on structural het- erogeneity have been found in other systems (for review, see Ref. 28). In support of this notion, we have never seen sheets of plasma membrane associated with vesicles in our fractions as they are in the intact cell. In fact, small vesicles are present at lighter densities in the gradient than the sheets (data not shown). Although we have no evidence that these vesicles arise from caveolae in the intact tissue, it is conceivable that the phosphorylation reaction may take place on such vesicles because they would represent a small subfraction (30% on morphological grounds (29)) of the total plasma membrane area and thus might not be expected to migrate with the bulk of the plasma membrane-associated enzymatic activity.

It has been suggested by a number of researchers that cGMP may play a role in the regulation of intracellular Ca2+ concentrations by either feedback (30,31) or feedforward (32) mechanisms. Since our data suggest that cGMP may control the state of phosphorylation of proteins intrinsic to the plasma membrane (or its caveolae), it may be involved in regulation of the flow of Ca2+ into and out of the cell rather than between intracellular compartments. Rasmussen et al. (32) have pro- posed a model in which cGMP exerts effects primarily on mitochondria and “microsomes” in response to the entry of “trigger” Ca2+ into the cell. Unless cGMP can be shown to

exert effects in these organelles, such models will have to be modified.

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THE OF BIOLOGICAL No. 8, of 1980 in Subcellular ... · Photoaffinity labeling with 8-N&*P]cIMP was performed using 25 to 150 pg of protein and incorporation of radioactive label into