ARCHIVES

OF

BIOCHEMISTRY

Phosphorylation

FRANK

AND

BIOPHYSICS

175, 321-331 (1976)

of a Low Molecular Na+,K+-ATPase

J. DOWD,

JR., BARRY

Weight Polypeptide Preparations1

J. R. PI’ITS,

AND

ARNOLD

in Beef Heart

SCHWARTZ

Department of Cell Biophysics, Division of Myocardial Biology, Baylor College ofMedicine and the FondrenBrown Cardiovascular Research and Training Center, The Methodist Hospital, and the Oral Disease Research Labomtory, Veterans Administration Hospital, Houston, Texas 77025 Received January 5, 1976 Sodium dodecyl sulfate-polyacrylamide gel profiles of a NaI-treated beef heart Na+,K+-ATPase preparation revealed the presence of two protein kinase substrates of low molecular weight, whereas a more purified citrate beef heart Na+,K+-ATPase preparation contained one low molecular weight polypeptide substrate. This enzyme preparation was phosphorylated in the presence of protein kinase, and phosphorylation was inhibited by protein kinase inhibitor. The phosphorylated product was identified as a phosphoester. Half maximal stimulation of protein kinase-catalyzed phosphorylation occurred at approximately 9 x lo-* M cyclic AMP. The low molecular weight (11,700) protein kinase substrate present in the heart preparations was eluted from polyacrylamide slab gels. The polypeptide fraction was reelectrophoresed and the polypeptide was removed from the gels, hydrolyzed, and analyzed for amino acid content. This polypeptide was different from other low molecular weight protein kinase substrates including troponin components, myosin light chains, and histones and is most likely of plasma membrane origin.

Several plasma membrane preparations from a variety of tissues are phosphorylated by cyclic AMP-dependent protein kinase. The membrane sources include kidney (l-31, liver (41, erythrocytes (5-71, the pituitary gland (8), heart (2, 91, skeletal muscle (lo), brain (2, ll-16), and fat cells (17). In a previous paper, we reported that cyclic AMP-dependent protein kinase catalyzed the phosphorylation of Na+,K+ATPase preparations from beef heart, beef brain, and dog kidney (2). Purification of Na+,K+-ATPase resulted in removal of endogenous protein kinase activity, but even in the highly purified dog kidney Na+,K+ATPase preparation a low level of phosphorylation catalyzed by exogenous protein kinase was observed. This phospho-

rylation occurred in the 93,000 molecular weight subunit of Na+,K+-ATPase (2). It has been postulated that membrane permeability changes result from phosphorylation of specific membrane-associated proteins in nerve tissue (181, kidney (31, toad bladder (19), and erythrocytes (20). In light of the evidence supporting a relationship between changes in plasma membrane characteristics and contraction of cardiac muscle, particularly with respect to calcium fluxes (21-271, we examined the phosphorylation of beef heart Na+,K+ATPase preparations in the presence of protein kinase. The emphasis was on low molecular weight polypeptides present in beef heart Na+,K+-ATPase preparations.

METHODS AND MATERIALS ‘This work was supported by USPHS Grant HL 07906, Contract NIH 71-2493, and American Heart Isolation of beef heart Na+,K+-ATPase. The Association Texas Affiliate, Houston Chapter. method used was that of Pitts and Schwartz (28). These studies represent partial fulfillment of the reThe fractions used in the study were the NaI-treated quirements for the Ph.D. degree for Frank J. Dowd, microsomal fraction and the final pellet from the Jr. deoxycholate treatment. _^_sodium citrate-sodium 3;zl

Copyright All righte

Q 1976 by Academic F’reee, Inc. of reproduction in any form reserved.

322

DOWD, PI’ITS AND SCHWAB’IZ

This pellet (citrate enzyme) as well as the NaItreated preparations were resuspended in 25 rnbr imidazoleHC1, 1 mM Tris-EDTA (pH 7.0) for the phosphorylation studies. Na’,K+-ATPase Assay. A spectrophotometric procedure was employed, utilizing a linked pyruvate kinase-lactate dehydrogenase system (29, 30). Each cuvette contained 2.5 rnbf MgCl*, 100 mM NaCI, 10 rnr.f KCI, 25 mM Tris-HC1 or histidine-HCl (pH 7.2), 2.5 mM Tris-ATP, 0.5 mM NADH (Sigma), 2.5 mM phosphoenolpyruvic acid (Sigma), and 0.02 ml of a combined pyruvate kinase-lactate dehydrogenase suspension (Sigma) at 37°C in a final volume of 2 ml. After a preincubation period of approximately 5 min. the reaction was initiated by the addition of enzyme. Phospkorylation studies. The method was described previously (2). The reactions, unless otherwise indicated, were carried out in 50 mM potassium acetate buffer (pH 6.2) containing 0.2 mM [ya*PIATP (New England Nuclear) (0.8 to 3 x lo6 cpm/ tube), 10 mM MgCl,, 10 rnxr KF, 2.5 my theophylline, 0.3 mM Tris-EGTA,Z 225-260 pg of Na+,K+ATPase, 20 pg of beef heart protein kinase (Sigma) in the presence or absence of 5 paa cyclic AMP, and in a total volume of 0.4 ml. The reaction was started by the addition of [y-“PIATP and incubated at 30°C for various time perioda. Routinely a preincubation period of 5 to 10 min was used. Termination of the reaction was accomplished by the addition of 3 ml of ice-cold 12.5% trichloroacetic acid (TCA) containing 1 mM Na,ATP (Sigma) and 1 mu potassium phosphate, followed by 0.2 ml of 0.63% bovine serum albumin. The centrifugation method was used to isolate the phosphorylated product. Pellets were washed three times by resuspension in 0.2 ml of 1 N NaOH followed by precipitation with 12.5% TCA containing 1 mM Na,ATP and 1 mM potassium phosphate. Gel electrophoresis. Gel electrophoresis in sodium dodecyl sulfate (SDS) was carried out according to a modified method of Uesugi et d. (31) and Shapiro et al. (32). For 7.5% gels, 10.1 ml of a filtered solution of 16.70% acrylamide and 0.45% bisacrylamide (BioBad), 11.25 ml of 0.1 M sodium phosphate (pH 7.1) containing 0.2% SDS, 1.125 ml of a fresh solution of 1.5% ammonium persulfate (Bio Bad) and 20 ~1 of N,N,N’N’-tetraethylmethylenediamine (TEMED; Matheson, Coleman and Bell) were used. For 10% gels, 10.1 ml of filtered solution of 22.20% acrylamide and 0.30% bisacrylamide was substituted for the above acrylamide solution. Protein samples were dissolved in 2% SDS, 1% 2mercaptoethanol (2ME; Bio-Bad) or 1 mM dithiothreitol (Bio-Bad) and 0.01 M sodium phosphate (pH 7.1) as described in the appropriate figure legends. The samples were either applied in 1520% glycerol directly to the gels or dialyzed against 0.1% SDS, 0.1% 2-ME in 0.01 M sodium phosphate (pH 7.1)

overnight at room temperature and applied to the gel in 15-2096 glycerol. The gel buffer and electrophoresis buffer were 0.05 M sodium phosphate containing 0.1% SDS (pH 7.1). Bromphenol blue (Eastman; 5 ~1 of a 0.15% solution) was used as a tracer dye. Electrophoresis was carried out with an applied current of 2 mA/gel for 15 min and then 5-6 mA/gel until the dye front was 1 cm from the bottom of the gel. A Buchler Model 1004 tube electrophoresis unit and a Heathkit Model IP-32 power supply were used. The gels were fixed, stained, and destained according to the method of Weber and Gsbom (33). Gels were scanned using a Model 2000 Gilford spectrophotometer with a Model 2410 linear transport atr tachment at 566 nm a&r staining. Gels containing radioactive samples were destained sutBc.iently to locate protein bands. These gels were sliced at& freezing on dry ice with a manifold of blades attached in a unit to give 2-mm gel slices and the slices were placed in scintillation vials. BBS-3 Fluoralloy TLA (Beckman) dissolved in toluene was added to each vial, after which they were counted in a Packard Model 574 Tricarb liquid scintillation spectrometer. Isolation of polypeptide substrates of protein kinose. Na+,K+-ATPase preparations were subjected to SDS-polyacrylamide gel (7.5%) electrophoresis as outlined above except that slab gels were used (Ortee, Model 4200). The gel slabs were 6.5 cm long x 10 cm wide x 0.3 cm thick. Prior to electrophoresis 5 to 8 mg protein in 1 to 2 ml of 0.1% SDS, 0.1% 2-ME, and 10 mM sodium phosphate (pH 7.1) prepared as above was layered in a solid layer on top of one gel slab. Two slabs were run at a time with a total of 50 mA applied until the tracking dye had penetrated the gel and then a total of lo&150 mA was applied. When the dye was approximately 1 cm from the end of the slab, the slab gels were removed by first loosening them with water injected around the slab with a syringe and needle and then gently pushing the gel out with a plastic sheet supplied with the unit. The gel was placed on a clean slab over a fluorescent light and the appropriate section was removed with a scalpel. The choice of sectioning was based on the calculated relative mobility (r.m.) of the phosphorylated peptide previously analyzed on analytical gels with the middle of the dye liont considered 1.0. Since the slab gels were not etained, the r.m. values were chosen with a certain latitude to insure removal of the desired bands (see Results). The band sections from four slabs (two runs) for each preparation were combined in a glass beaker and finely minced with a clean scissors. A small amount of 0.1% SDS, sutlicient to cover the slices, was added to the beaker and incubated at 37°C for 10 h according to the method of Weber and Osbom (33). The supematant was withdrawn and a second elution was performed at 37°C for 8 h. The two supernatants were combined and filtered to remove any remain-

PHOSPHORYLATION

OF A MEMBRANE

ing acrylamide and the solutions were lyophilized to dryness. Deionized water was added to the residues to give an approximate SDS solution of 1% and the SDS extracted twice with 9 vol of ice-cold acetone. The protein precipitate was taken up in a small portion of 1 rnM Tris-EDTA (pH 7.0) and dialyzed against 106 vol of 1 rnM Tris-EDTA (pH 7.0) for 4 days with five changes of buffer. The suspensions were then frozen until used. Amino acid analysis. The peptides eluted from the slab gels were tested for phosphorylation in the presence of protein kinase. In addition, they were subjected to further electrophoresis on polyacrylamide gels in the presence of 0.1% SDS. The electrophoresis, including prior solubilization and dialysis, was performed as outlined above, except that 10% polyacrylamide analytical gels were used. From 30 to 120 pg of protein was applied to each gel. Electrophoresis, staining, and destaining were performed as above. All equipment, including the destainer, was thoroughly cleaned before use. Representative tubes were scanned and molecular weights reeetimated using ovalbumin (43,000), pepsin (35,000), trypsin (23,300), myoglobin (17,200), and cytochrome c (11,700) as standards. The desired visualixed bands were removed by cutting them out with a new scalpel blade. The method for hydrolysis was a modification of that of Houston (34). Five to six gels were run for each peptide sample. All gel samples (approximately 0.5-1.2 cm in length) for each peptide were combined in a hydrolysis tube (Phoenix Precision Instruments) and 100 ~1 of 2-ME was added followed by 5 ml of 6 N HCl. The samples were mixed with a vortex mixer and quick-frozen in a dry ice-ethanol bath and a vacuum was applied to the tubes via a fitted glass attachment with a stopcock. The vacuum obtained was approximately 0.007 mm Hg employing a dry ice-ethanol trap. The stopcock at this point was closed to retain the vacuum and the tubes incubated at 110 2 3°C for 22 h. The sample tubes were then cooled in a refrigerator after removing the vacuum and centrifuged for 10 min to pack the murky white residue. The supematants were removed and 2 ml of 1 N HCl was added. Each sample was shaken and heated in an oven at 100°C for 15 min followed by cooling and centrifugation. Tbe supematants were removed and the extraction procedure was repeated one more time. The supernatants were then combined and evaporated to dryness in a flash evaporator at 40°C. A small aliquot of sodium citrate buffer (pH 2.20 * 0.02) was added so that 500 ~1 of the sample contained the equivalent of 0.3 to 3 nmol of the original polypeptide. The buffer was composed of 67 mM sodium citrate with 48.6 mM thiodiglycol (Pierce Chemical Co.) and 1 rnxf octanoic acid (Pierce Chemical Co., Sequenal Grade) added as antimold agents. The pH was adjusted to 2.20 + 0.02 with concentrated HCl. Amino acid analyses were performed on a Beck-

323

POLYPEPIIDE

man Model 116 Amino Acid Analyzer with expanded scale capability (35). The short column was 0.9 x 15 cm to ensure the adequate removal of ammonia from the system with no overlap of the ammonia and hi&line peak. (The Ninhydrin line was switched to drain when the ammonia peak was recording.) Elution buffers were the same as the buffer used to dissolve the amino acid samples except that the pH used for the elution of acidic and neutral amino acids was 3.25 2 0.02 and 4.30 f 0.02, respectively. The buffer used to elute basic amino acids was the same except that it contained 0.117 M sodium citrate at a pH of 5.25 -c 0.02. Only the pH 3.25 buffer contained thiodiglycol (24.3 mM). Quantitation of amino acids was performed by estimation of peak areas either by calculation of half the peak height times the peak width or with a planimeter (Gelman Instrument Co.). Isolation

of crude

protein

kinnse

inhibitor.

The

method was a modification of the procedure described by Walsh et al. (36). Approximately 1809 g of rabbit skeletal muscle was homogenized in 2.5 vol of 4 mu Tris-EDTA (pH 7.0). The homogenate was centrifuged at 1290~ for 30 min. The supematant was decanted through glass wool and heated quickly in small aliquots to 90°C. The mixture was cooled and filtered through glass wool. Precipitation of the supematant with 15% trichloroacetic acid, resuspension of the pellet, and dialysis were carried out according to the method of Walsh et al. (36), except that potassium phosphate buffer was used throughout. Estimation ofprotein concentrations. Protein was estimated by the method of Lowry et al. (37). RESULTS

The NaI-treated beef heart enzyme phosphorylated in the presence of exogenous protein kinase revealed polyacrylamide gel profiles in which several protein bands were present, but the majority of the label was incorporated into two low molecular weight polypeptides (Fig. 1). Specific activities of these Na+,K+-ATPase preparations were 15 to 20 pmol/Pi/mg of protein/h. Upon further purification using the sodium citrate-DOC? method, removal of the larger of the two polypeptides was accomplished (Fig. 2). This preparation, therefore, contained one principle protein kinase substrate and is referred to as “the 2Abbreviations used: EGTA, ethylene glycol-bis(P-aminoethyl etherW,N’-tetraacetate; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; DOC, deoxycholate; TEMED, NJVfl’JV’tetraethylmethylenediamine; 2- ME, 2 - mercaptoethanol; r.m., relative mobility.

324

DOWD

POLYACRYLAMIDE

GEL

LENGTH

PITTS AND SCHWARTZ

ml082 IMM)

FIG. 1. Protein kinase-catalyzed phosphorylation of NaI-treated beef heart Na+,K+-ATPase: polyacrylamide gel profile. The Na+,K+-ATPase was isolated according to procedures given in the Methods section. Phosphorylations were conducted for a 30-min incubation period in the presence and absence of 5 PM cyclic AMP as described in Methods except that potassium phosphate (pH 6.5), 480 wg of Na+,K+-ATPase, and 40 pg of protein kinase (Sigma) were used. The final ATP concentration was 20 PM (12.7 @i/tube). The reaction was stopped by adding 3 ml of 10 mM potassium phosphate (pH 6.5) at 4”C, and 100 ~1 of 500 mM Tris-CDTA, [(1,2cyclohexylenedinitrilo) tetraacetic acid]. The tubes were kept on ice for 5 min and centrifuged at 150,OOOgfor 15 min. The supernatant was removed and the pellets were resuspended in 3 ml of 10 rnxr potassium phosphate with 100 ~1 of 500 mM TrisCDTA and recentrifuged. Resuspension and centrifugation were repeated one more time. Electrophoresis was carried out as described in Methods except that the pellets were solubilized in 150 ~1 of 10 mM sodium phosphate (pH 7.1), 5% SDS, and 10 rnM 2ME and dialyzed against 10 mM sodium phosphate @H 7.1) containing 0.1% SDS and 1 mM dithiothreitel. The samples were then heated to 78°C for 30 min, incubated at 37°C for 3 h, and applied to the gels in lo-p1 aliquots with glycerol and tracking dye. Electrophoresis was carried out in 7.5% polyacrylamide and 0.1% SDS. Further details are given in Methods. The arrows as well as the relative mobility value on the abscissa are explained in the text.

citrate enzyme” (28). Control phosphorylation experiments were carried out in which Na+,K+-ATPase was added after the cessation of phosphorylation and coelectrophoresed with protein kinase, confuming the fact that the Na+,K+-ATPase preparation was the source of the phosphorylated polypeptide. The heart citrate enzyme preparation was thought to be useful for studying membrane phosphorylation since it was phosphorylated in the presence of protein kinase, the phosphorylation was stimu-

lated by cyclic AMP, the majority of 32P incorporation in the preparation was into one polypeptide, and the polypeptide appeared to be an authentic membrane component. Since large quantities of the purifled polypeptide were not available, we examined certain characterisitcs of its protein kinase-catalyzed phosphorylation by studying the phosphorylation of the beef heart citrate enzyme preparation. The absence of substantial protein kinase activity in the citrate enzyme preparation is shown in Table I. In the presence of histone, neither the Na+,K+-ATPase preparation nor cyclic AMP added to the Na+,K+-ATPase preparation was associated with significant stimulation of phosphorylation. The pH profile of protein kinase-catalyzed phosphorylation of a heat-denatured Na+,K+-ATPase preparation (boiled for 20 min to eliminate enzyme activity) revealed a pH optimum in the presence of cyclic AMP of approximately 7.4 (Fig. 3). The pH optimum in the absence of cyclic AMP may not have been reached but was highest at pH 8.0 for the various pH values tested (Fig. 3). The pH profile is apparently affected by cyclic AMP. Linearity of phosphorylation was observed in the enzyme concentration range tested (Fig. 4 and 5).

0 ( GEL

LENGTH

(mm)

rm074

2. Protein kinase-catalyzed phosphorylation of beef heart citrate Na+,K+-ATPase: polyacrylamide gel profile. All procedures were conducted as described in the legend to Fig. 1 except that phosphorylations were performed at pH 6.0 and 270 pg of Na+,K+-ATPase was used (original sp act 385 pmol Pi/mg/h). The final ATP concentration was 20 PM (7 &i/tube). The cyclic AMP concentration was 5 P.M. Samples were not dialyzed prior to electrophoresis. Arrows and relative mobility value are explained in the text. FIG.

PHOSPHORYLATION TABLE

OF A MEMBRANE

I

ABSENCE OF END~GENOUS PROTEIN KINASE ACTIVITY IN CITRATE BEEF HEART Na+,Kf-ATPase”

Na+,K+-ATPase histone

Na+,K+-ATPase (-)cAMP Lob 4.3

(-)cAMP

(+) CAMP 6.9 5.8

+

(+)cAMP

4.7 3.6

4.5 4.8

U Phosphorylations were carried out as in Methods except that a pH of 7.15 (Tris-HCI) was used along with 150 pg of Na+,K+-ATPase and 666 pg of histone (type II-A calf thymus) where indicated. A 3min incubated period was used. Numbers represent individual values from separate phosphorylations. The cyclic AMP concentration was 5 PM. b Picomoles of 32P incorporated.

325

POLYPEPTIDE

rylation of the Na+,K+-ATPase preparation was sensitive 4x1protein kinase inhibitor (absence of inhibitor = 26 pmol; presence of inhibitor = 3 pmol), and the effect was seen only on the cyclic AMRstimulated portion of phosphorylation. Activation of protein kinase by cyclic AMF’ using the citrate enzyme beef heart Na+,K+-ATPase preparation as substrate was observe6 over a range of concentrations with half maximal stimulation oc500 2 z 400 &F gE2 0. a 300 I

t

CAMP

/

300 -

. t CAMP

8 k 200 f?

[~OTEIN

g

l

a I

-

z g a

100

:r

Do ,’

-CAMP ,co-o/-’

P’

SD o’s’ 0’

50

6.0

70

SO

PH

FIG. 3. Effect of pH on protein kinase-catalyzed phosphorylation of beef heart Na+,K+-ATPase. Citrate beef heart Na+,K+-ATPase (original sp act 341 pmol Pi/mg/h) was denatured by heating at 100°C for 20 min. The buffer used was Gomori’s Trismaleate. The final fluoride concentration was 5 mM. Sodium concentrations in the buffer were made equal at each pH so that the final assay concentration in every case was 42 mM. Tubes contained 280 pg of Na+,K+-ATPase and 20 pg of beef heart protein kinase (Sigma). Conditions were otherwise the same as described in Methods. Individual values are means of triplicates. Corrections have been made for phosphorylation in the presence of protein kinase alone at each pH. The cyclic AMP concentration was 5 &CM.

The majority of the phosphorylated product was of the phosphoester type since it was unstable in alkali, stable in acid, and hydroxylamine insensitive (2). The protein kinase-catalyzed phospho-

25 wnsE&g

50 /ml)

FIG. 4. Effect of protein kinase concentration on the protein kinase-catalyzed phosphorylation of beef heart Na+,K+-ATPase. Citrate beef heart Na+,K+ATPase (138 pg) was used with the various concentrations of beef heart protein kinase (Sigma). The Na+,K+-ATPase (original sp act 312 wmol Pi/mg/h) was denatured by heating as described in the legend to Fig. 3. Other conditions were as outlined in Methods. Individual values are the means of duplicates. Correction has been made for phosphorylation in the presence of protein kinase alone in the presence or absence of cyclic AMP (5 PM).

FIG. 5. Time course of protein kinase-catalyzed phosphorylation of beef heart Na+,K+-ATPase. The conditions used were those described for Fig. 4 and in Methods except that 5 pg of protein kinase was used and the incubation times were varied as indicated. The cyclic AMP concentration was 5 FM.

326

DOWD, PITIS

curring at approximately 9 x lOa M (Fig. 6). The beef heart citrate enzyme preparation and the NaI-treated preparation were subjected to slab gel electrophoresis and the specific gel segments were excised to elute the desired polypeptide. The sections of gels removed were those corresponding to the areas between the arrows in Fig. 1 and 2. The section denoted in Fig. 1 is referred to as the “lower molecular weight phosphorylated polypeptide” from the NaI preparation. After extraction and removal of SDS, the polypeptide fractions were phosphorylated by protein kinase. Each fraction showed marked stimulation of phosphorylation in the presence of cyclic AMP (Table ID. These substrates were then subjected to SDS-polyacrylamide gel electrophoresis after phosphorylation. Gels were stained and destained. Figure 7 identifies the phosphorylated substrates in the beef heart citrate enzyme preparations and NaI-treated preparation. Cyclic AMPstimulated phosphorylation was observed in a single band in the citrate enzyme preparation (Fig. 7A) and in two bands for the NaI-treated preparation (Fig. 7B). Not all of the radioactivity coincided with the protein peaks at or near M, 11,700 in Fig. 7B. The radioactive peak of lesser mobility could possibly be due to contamination by the larger of the two phosphorylated poly-

h

L

l’;9s

..-log

CAMP

_ i .6

CONCENTRATION

.-M 5

4

3

IMI

FIG. 6. Effect of cyclic AMP concentration on the protein kinaae-catalyzed phosphorylation of beef heart Na+,K+-ATPaee. Citrate beef heart Na+,K+ATPase (original ep act 318 pm01 Pi/mg/h) wae denatured as described for Fig. 3. Each tube contained 136 pg of denatured Na+,K+-ATPaae and 5 rg of beef heart protein kinaee (Sigma). Phoaphorylations were carried out for 3 min. Correction hae been made for phcephorylation in the presence of protein kinaee alone. Other conditione were described in Methods. Values are means 2 SEM.

AND SCHWARTZ TABLE PROTEIN KINASIJ-CATALYZED POLYPE~TIDB FRACTIONS

II PHO~PHORYLATION

OF

WOY Na+,K+-ATPaae Preparations” Citrate Beef Heart EnNaI-Beef Heart “lower fraction” zyme - CAMP nob 259

+ CAMP 1180 1580

- CAMP 227

+ CAMP 1100

a Individual fractions were those removed from 7.5% slab gels illustrated in Figs. 1 and 2. Phosphorylations were performed for 15 min as in Methods with 20 Fg of beef heart protein kinase (Sigma). The substrate protein used in each case was: citrate beef heart enzyme, 27 pg and NaI-beef heart “lower fraction”, 33.5 pg. Numbers are individual values and are corrected for phosphorylation in the absence of substrate. h Picomoles of 32P incorporated per milligram of substrate protein.

peptides present in the original NaI-preparation (Fig. 1) and removed from the slab gels during the extraction procedure. In addition, the protein extracted from the NaI-treated preparation appeared as two protein bands in Fig. 7. This suggests that the protein region extracted was not composed of just one polypeptide, as might be expected due to the relatively crude nature of the original Na+,K+-ATPase preparation used. This could also account for the difference in relative composition of amino acids when comparing the M, 11,700 polypeptide extracted from the citrate enzyme preparation to that extracted from the NaI preparation (Table Iv). In preparation for amino acid analysis, gel segments containing the polypeptides from both Na+,K+-ATPase preparations (unphosphorylated) were removed after a separate series of electrophoresis. The gel sections removed are denoted by brackets in Fig. 7. Each is designated by a letter with the corresponding calculated molecular weight given in the legend for Fig. 7. Since the gels were stained and destained, the desired bands could readily be seen and removed. The amino acid analysis of the fraction in Fig. 7 is presented in Table III. TO assign numbers of residues per molecule, histidine was set at 1.0 since its occurrence was the least of those tested. This multiple

PHOSPHORYLATION

40 GEL

60

LENGTH

OF A MEMBRANE

SO (+I Imm)

FIG. 7. SDS-polyacrylamide gel electrophoresis of phosphorylated low molecular weight polypeptide protein kin&se substrates from beef heart Na+,K+ATPase. Polypeptide fractions (gel segments between arrows in Fig. 1 and 2) were eluted from 7.5% slab polyacrylamide gels followed by lyophilixation, acetone extraction, and dialysis as described in Methods. Phosphorylation of these fractions was performed with 20 pg of protein kinase in the presence (hatched area) or absence (solid area) of 5 PM cyclic AMP at pH 6.5 (potassium phosphate buffer). Reactions were stopped with 3 ml of 12.5% TCA containing 1 mre Na,ATP and 1 mM potassium phosphate. The tubes were mixed on a vortex mixer and the contents were dialyzed against 1000 vol of deionized water overnight at 0°C. The content of each dialysis bag was lyophilixed and taken up in 100 ~1 of 10 mu sodium phosphate (pH 7.1), 1% SDS, and 1% 2-ME. These were then heated at 78°C for 15 min and glycerol and bromphenol blue added. The fractions were applied to 10% polyacrylamide gels containing 0.1% SDS in 66~1 aliquots. Electrophoresis in cylindrical gels was performed as described in Methods. (A) Low molecular weight polypeptide from citrate beef heart Na+,K+-ATPase (see Fig. 2) using 36 pg for phosphorylation; (B) lower molecular weight phosphorylated polypeptide band from NaI beef heart Na+,K+-ATPase preparation (see Fig. 1) using 100 pg for phosphorylation. Areas denoted by brackets in A and B correspond respectively to the polypeptides “A” and “B” removed from the gels for amino acid analysis. The calculated molecular weight in each case was 11,700.

was the only choice because of the molecular weight of the polypeptide. The amino acids analyzed accounted for 81.1% of the total molecular weight. Since sulfur-containing amino acids as well as tryptophan are destroyed by acid hydrolysis (34, 38). the values for these amino acids were not estimated. A comparison was made of molecular weights and amino acid compositions of troponin components from skeletal muscle

327

POLYPEPTIDE

and myosin light-chain components from heart muscle with the polypeptide from the beef heart citrate Na+,K+-ATPase preparation (Table IV). The myosin lightchain and troponin components are distinguished from this polypeptide on the basis of molecular weight and major. differences in amino acid ratios. This does not rule out the possibility that the polypeptides could result from proteolytic breakdown of troponin, myosin, or other protein, but the care exerted to denature the protein preparations by heating makes this possibility remote. TABLE III AMINO ACID ANALYSIS OF Low MOLECULAR POLYPEPTIDE

FRACTIONS

Na+,K+-ATPase Amino acid

FROM

WEIGHT BEEF HEART

Preparations” “A”

Residues Weight/ weight per molecule % Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Total

5.7 1.0 5.2 9.0 4.7 5.3 12.7 3.8 5.5 8.6 5.8 2.6 10.1 2.1 3.7

6.2 1.2 6.9 8.9 4.0 3.9 14.0 3.2 2.7 5.3 4.9 2.5 9.8 2.9 4.7

(=

“B”

ixed to “A”) 6.2 0.8 5.1 8.6 5.0 6.1 12.7* 7.9 14.6 8.5 4.9 3.2 8.1 1.4 4.8

81.1%

“Amino acid analysis was performed as described in Methods. Fraction designations “A” and “B” are explained in the legend to Fig. 7. In Fraction “A,” histidine was made equal to 1 residue/molecule. A higher multiple of histidine residues/molecule gave a combined amino acid weight value above the calculated molecular weight of the; polypeptide (11,700). The asterisk indicates the amino acid with which the “B” fraction was normali$ed to the “A” fraction. The choice of the amino acid used for normalization was based on the amino acid which gave nearly minimum percentage difference (for each amino acid) between “B” and “A.” This was done to facilitate comparison of amino acid profiles between polypeptides. The combined weight of the “A” fraction amino acids listed (in peptide linkage) is 9494.9.

328

DOWD,

PITTS

AND

TABLE COMPARISON Amino

acid

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine

OF AMINO

“A”

5.7 1.0 5.2 9.0 4.7 5.3 12.7 3.8 5.5 8.8 5.8 2.6 10.1 2.1 3.7

SCHWARTZ IV

ACID RESIDUES FROM “A” TO TROP~NIN MYOSIN LIGHT CHAIN COMPONENT@

Proteolipid sarcoplasmic reticulum 1.1 0.9 10.0 5.1 6.9 8.2 10.9 2.2 2.9 3.3 6.2 4.0 17.1 7.1 4.9

Troponin

Components

(TnI)

Cfi’I’)

(TnC)

26.4 4.8 17.0 19.3 5.3 12.6 40.7 8.5 11.5 18.8 8.5 6.4 22.3 2.8 3.7

51.8 12.2 30.2 40.0 14.4 29.7 108.4 19.4 27.5 45.9 14.9 21.2 30.2 5.0 7.2

9.9 0.3 5.4 17.5 4.9 5.1 24.0 0.9 11.9 12.9 5.1 6.0 7.5 1.7 8.3

COMPONENTS

AND

Myosin light-chain components “17,000” “26,000” 16.9 1.4 3.5 18.6 7.5 3.2 20.7 7.7 8.9 12.0 7.8 7.7 10.1 1.6 10.1

29.2 3.0 5.3 21.4 12.2 6.1 33.2 20.8 16.9 21.8 11.8 9.1 17.8 2.9 10.8

a Numbers listed are number of residues per molecule. The values for myosin light chains of beef heart muscle are taken from Katagiri and Morkin (44). Troponin amino acid residues per molecule are calculated from the data of Staprans et al. (52, Table II) using the listed molecular weights for each component. The amino acid residues of the sarcoplasmic reticulum proteolipid are taken from MacLennan et al. (60). DISCUSSION

The citrate enzyme from beef heart offered the advantage of a Na+,K+-ATPase preparation which possessed a protein substrate which accounted for almost all of the phosphate incorporation catalyzed by protein kinase. Phosphorylation occurred almost totally in what appeared to be one polypeptide. This preparation showed phosphorylation characteristics similar to other Na+,K+-A’ITase preparations tested (2).

The pH optimum in the absence of cyclic AMP, however, was quite different from that in the presence of cyclic AMP. This may reflect a cyclic AMP effect on the pH optimum exhibited by protein kinase for this substrate. Other investigators have reported higher pH optima for protein kinase-catalyzed phosphorylations in the absence of cyclic AMP than in its presence (5, 39). Weller and Kodnight (161, for example, found a broad very flat pH profile (from pH 7.0 to 8.0) for intrinsic kinase activity in ox brain preparations. Waddy and MacKinlay (40) reported a high pH optimum for phosphorylating reactions of lactating bovine mammary glands. The present studies took advantage of

the fact that specific polypeptides eluted from slab polyacrylamide gels, although they had been boiled and treated with SDS and acetone, could still serve as substrates for protein kinase, a phenomenon that is characteristic of protein kinase substrates (3, 10). Isolation of corresponding low molecular weight protein kinase substrates from the two beef heart Na+,K+-ATPase preparations revealed similarities in amino acid content. These similarities were expected for the lower molecular weight protein kinase substrate in the NaI-treated heart preparation and the single low molecular weight substrate in the heart citrate enzyme preparation since these polypeptides appeared to be identical. Some differences were observed, however, in the amino acid profiles and this may have been due to contamination in the less purified NaI preparation. The polypeptide had a molecular weight of 11,700 on SDS-polyacrylamide gel electrophoresis (10%). A comparable polypeptide was observed in brain and it too had a similar amino acid profile (data not shown). Analysis of this gel region of the citrate enzyme indicated the presence of PAS-positive staining material

PHOSPHORYLATION

OF A MEMBRANE

suggesting that this protein kinase substrate is a glycoprotein (data not shown). The results of Krause et al. (9) indicate the presence of a glycoprotein in the comparable gel region of their NaI-treated pig heart preparation. Chemical characterization of the phosphorylated products in the Na+,K+-ATPase preparations tested indicate the predominance of phosphoesters. Furthermore, the ability to phosphorylate the polypeptide previously extracted from SDS gels indicates that the substrate is, in all likelihood, protein in nature. This is an important consideration because of the proximity of the 11,700 molecular weight polypeptide to the dye front with possible contamination of the gel region by [32P]ATP or [32P]Pi. Although the fairly extensive purification of the Na+,K+-ATPase probably eliminated all nonmembrane components, it was advisable to rule out the possibility that this polypeptide could be either the cardiac myosin light chain which has a molecular weight of 17,000-19,000 (44, 451, or TnI, a component of troponin which has a molecular weight of 23,000 (52), both of which are substrates for protein kinases (41-45 and 46-51, respectively). It is clear from the amino acid composition that this polypeptide is neither a component of troponin nor a myosin light chain. It is also readily distinguishable from histones (5355). The level of protein kinase-catalyzed phosphorylation of the low molecular weight polypeptide is much higher than that observed for the 93,000 molecular weight subunit of Na+,K+-ATPase. We previously reported that this subunit was phosphorylated in the presence of protein kinase and this phosphorylation was stimulated by cyclic AMP (2). Comparison of the phosphorylation levels of the purified Na+,K+-ATPase to the low molecular weight polypeptide of beef heart demonstrates that the phosphorylation levels attained for the purified Na+,K+-ATPase from dog kidney catalyzed by exogenous protein kinase in the presence of cyclic AMP was approximately 27 pmol/mg protein (2, Table I), whereas in the present

POLYPEPTIDE

329

study the levels were between 1190 and 1580 pmol/mg protein. The conditions used for each phosphorylation were similar although not identical. For the dog kidney Na+,K+-ATPase preparation a 30-min incubation period was used whereas 15 min was used in the phosphorylation of the low molecular weight polypeptide reported in the present study. In addition, the value reported for the kidney preparation is in terms of milligrams of protein for the two subunit enzyme, whereas in the beef heart preparation of the present study the values given are in terms of milligrams of the extracted polypeptide. Based on the published stoichiometry of the two subunits of purified dog kidney Na+,K+-ATPase, however (56,57), the total protein kinase-catalyzed phosphorylation for the subunit would be less than 45 pmol/mg protein of the 93,000 molecular weight subunit. Thus, the low molecular weight polypeptide of the heart preparation is a quantitatively more effective substrate for protein kinase than the catalytic subunit of Na+,K+-ATPase of the kidney. This also can be observed in the heart preparation by comparing the lower level of phosphorylation found in the area of the corresponding 93,000 molecular weight region of the polyacrylamide gels to that seen in the region of 11,700 molecular weight polypeptide. In fact, demonstrating phosphorylation of the 93,000 molecular weight subunit is most difficult except in the purified Na+,K+-ATPase preparation. The physiological significance of the protein kinase-catalyzed phosphorylation of the low molecular weight polypeptide is not clear but Will et al. (58) have reported that phosphorylation of a NaI-treated Na+,K+-ATPase preparation from pig heart by an intrinsic protein kinase stimulated calcium binding to the membranes increasing the binding constant for calcium fourfold. We have been unable to repeat this work, as yet. This could be due to differences in the membrane preparations. Gibson and Newcomb (591, in a preliminary report, have recently reported incorporation of 32P into a gel band with a molecular weight of approximately 10,000

330

DOWD,

PITTS

from a rat myocardial microsomal preparation. The phosphorylation was catalyzed by a protein kinase endogenous to the membrane fraction but the phosphorylation was not stimulated by cyclic AMP. Their findings are in other respects similar to those reported here. The lack of effect of cyclic AMP may have been due to the different source of protein kinase. While we cannot yet define the function of this polypeptide, it could possibly fulfll a similar role to that proposed for the “proteolipid” component of sarcoplasmic reticulum first described by MacLennan et al. (601. Recently, Racker and Eytan (61) have shown that when this proteolipid is incorporated into liposomes together with the purified Ca2+-A!I’Pase protein, it increases the rate of calcium transport into the liposomes fourfold but has no effect on the rate of ATP hydrolysis. Consequently, the efficiency of the calcium pump is greatly increased. It seems possible that the polypeptide described here could play a similar role in regulating the efficiency of the sarcolemmal sodium pump. This raises the intriguing possibility that phosphorylation of this polypeptide by protein kinase could regulate the efficiency of the sodium pump even though it has no effect on Na+,K+-ATPase activity in vitro. We acknowledge the helpful discussions and assistance of Drs. M. L. Entman, Y. S. Reddy, and R. P. Feller during the course of this investigation. REFERENCES

phys. 2. Dcm,

W-T. H., WALKENBACH, K. H. (1972) B&hem. Res. Commun. 49,1510-1517. F. J., AND SCHWARTZ, A. (1975) J.

Cell. Cardiol. 3. DOU~A, (1972) 4. SHLATZ,

H.

P.,

SCHWARTZ

Biol. Chem. 249, 323-328. 9. KRAUSE, E. G., WILL, H., PELOUCH, V., AND WOLLENBERGER, A. (1973) Acta. Biol. Med. German. 31,37-43. 10. SULAKHE,

P. V.,

AND DRUMMOND, G. I. (1974) Biophys. 161,448-455. 11. ALEXANDER, D. R., AND RODNIGHT, R. (1974) Biochem. J. 137, 253-262. 12. EHRLICH, Y. H., AND RQ~TENBERG, A. (1974)

Arch. B&hem.

FEBS Lett. 45,237-243. 13. JOHN~~N, E. M., UEDA, T., MAENO, H., AND GREENGARD, P. (1972) J. Biol. Chem. 247, 5650-5652. 14. UEDA, T., MAENO, H., AND GREENGARD, P. (1973) J. Biol. Chem. 248, 8295-8305. 15. WELLER, M., AND RODNIGHT, R. (1971) B&hem. J. 124,393-406. R. (1973) Biochem. 16. WELLER, M., AND RODNIGHT, J. 132,483492. 17. CHANG, K. J., MARCUS, N. A., AND CUATRECASAS, P. (1974) J. Biol. Chem. 249,6854-6865. 18. GREENGARD, P. (1973) in Protein Phosphorylation in Control Mechanisms, Miami Winter Symposium (Huijing, F., and Lee, E. Y. C., eds.) Vol. 5, p. 145, Academic Press, New York. R. J., WALTON, K. G., CURRAN, P. 19. DELORENZO, F., AND GREENGARD, P. (1973) Proc. Nat.

Acad. Sci. USA 70, 880-884. 20. RUWLPH,

S. A.,

AND GREENGARD,

P. (1974)

J.

Biol. C&m. 249, 5684-5687. 21. BAILEY,

L. E., AND DRESEL,

P. E. (1968)

J. Gen.

Physiol. 52, 969-982. 22. BAILEY,

L. E., AND SURES, H. A. (1971)

J. Phur-

macol. Exp. Ther. 178, 259-270. G. A., AND FRANK, J. S. (1972) J. Cell Biol. 54, 441-455. 24. LANGER, G. A. (1972) Circulation 46,180-187. III25. LANGER, G. A. (1974) Circ. Res. 35, suppl., 23. LANCER,

ACKNOWLEDGMENTS

1. FORTE, L. R., CHAO, J., AND BYINGTON,

AND

R.

BioMol.

7, 483-497. SANW,

H., AND HECHTER, 0. 91,757-763. L., AND MARINETTI, G. V. (1971) Bitthem. Biophys. Res. Commun. 45, 51-56. 5. GUTHROW, C. E., ALLEN, J. E., AND RASMUSSEN, H. (1972) J. Biol. Chem. 247, 8145-8153. 6. ROSES, A. D., AND A~PEL, S. H. (1973) J. Biol. Chem. 248, 1408-1411. 7. RUBIN, C. S., AND F~SEN, 0. M. (1973)Biochem.

Endocrinology

Biophys. Res. Commun. 50,421-429. 8. LEMAY, A., DE~CHENES, M., LEMAIRE, S., POIRIER, G., POULIN, L., AND LABIUE, F. (1974) J.

91-H-I-98. 26. NIEDERGERIKE, R. (1963) J. Physiol. 167,515-580. 27. REUTER, H., AND BEELER, G. W. (1969) Science 163, 399401. 28. PITTS, B. J. R., AND SCHWARTZ, A. (1975) Biochim. Biophys. Acta 401, 184-195. 29. ALBERB, R. W., AND KOVAL, G. J. (1962) Life Sci. 1, 219-222. S. 30. SCHWARTZ, A., ALLEN, J. C., AND HARIGAYA, (1969) J. Pharmucol. Exp. Ther. 168,31-41. N. C., DIXON, J. F., HEXUM, 31. UESUGI, S., Dm~x, T. D., DAHL, J. L., PERDW, J. F., AND HOKIN, L. E. (1971) J. Biol. Chem. 246, 531-543. 32. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V., JR. (1987) B&hem. Biophys. Res. Commun. 28, 815-820. M. (1969) J. Biol. 33. WEBER, K., AND OSBORN,

Chem. 244,44064412. L. L. (1971)AnaZ. 34. HOUSTON, D. H., STEIN, 35. SPACEMAN,

B&hem.

44,81-88.

W. H., AND MOORE,

S.

PHOSPHORYLATION

OF

A MEMBRANE

(1958) Anal. Chem. 30, 1190-1266. 36. WALSH, D. A., ASHBY, C. D., GONZALEZ, C., CALIUNS, D., FISCHER, E. H., AND KREBS, E. G. (1971) J. Biol. Chem. 246, 1977-1985. 37. Loway, 0. H., ROSEBROUGH, N. J., FAI~R, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 38. BLACKBURN, S. (1968) Amino Acid Determination: Methods and Techniques, p. 127, Marcel Dekker, New York. 39. CORBIN, J. D., BROSTROM, C. O., ALEXANDER, R. L., AND KREBS, E. G. (1972) J. Biol. Chem. 247, 3736-3743. 40. WADDY, C. T., AND MACKINLAY, A. G. (1971) Biochim. Biophys. Actu 250, 491-500. 41. PERRIE, W. T., SYILLIE, L. B., AND PERRY, S. V. (1973) Biochem. J. 135, 151-164. 42. PERRIE, W. T., SMILLIE, L. B., AND PERRY, S. V. (1972) Cold SpringHarbor Symp. Quant. Biol. 37, 17-18. 43. PIRES, E., PERRY S. V., AND THOMAS, M. A. W. (1974) FEBS Lett. 41, 292-296. 44. KATAGIRI, T., AND MORKIN, E. (1974) Biochim.

Biophys. Acta 342, 262-274. N., ANDPERRY, S. V. (1975)Biochem. J. 151, 99-107. 46. BAILEY, C., AND VILLAR-PALAI, C. (1971) Fed. Proc. (Fed. Amer. Sot. Exp. Biol.) 30, 1147

45. FREAREKIN,

(Abstract). 47. REDDY, Y. S., BALLARD, SCHWARTZ, A. (1973) 461-471.

D.,

Gnu,

N.

Y.,

AND

J. Mol. Cell. Cardiot. 5,

331

POLYPEPTIDE

S. V., AND COLE, H. A. (1974) B&hem. J. 141, 733-743. 49. PERRY, S. V., AND COLE, H. A. (1973) B&hem. J. 131,425-428.

48. PERRY,

50. STULL, J. T., BROSTROM, C. O., AND KREBS, E. G. (1972) J. Biol. Chem. 247, 5272-5274. 51. COLE, H. A., AND PERRY, S. V. (1975) B&hem.

J. 149, 525-533. 52. STAPRANS, I., TAKAHASHI, H., RUSSEL, M. P., AND WATANABE, S. (1972) J. B&hem. 72,723735. 53. DELANGE, R. J., SMITH, E. L., FAMBROUGH, D. M., AND BONNER, J. (1968) PFOC. Nat. Acad. Sci. USA 61, 1145-1146. 54. IWAI, K., ISHIKAWA, K., AND HAYASHI, H. (1970) Nature (London) 226, 10561058. 55. STARBUCK, W. C., MAURITZEN, C. M., TA~OR, C. W., SAROJA, I. S., AND BUSCH, H. (1969) J.

Biol. Chem. 243, 2038-2047. 56. LANE, L. K., COPENHAVER, J. H., JR., LINDENMAYER, G. E., AND SCHWARTZ, A. (1973) J. Biol. Chem. 248, 7197-7200. 57. KYTE, J. (1972) J. Biol. Chem. 247,7642-7649. 58. WILL, H., S~HIRPKE, B., AND WOLLENBERCER, A. (1973) Acta Biol. Med. German. 31, 45-52. 59. GIBSON, K., AND NEWCOMB, M. (1975) B&hem.

Sot. Trnns. 3, 547-548. 60. MACLENNAN, SEEMAN,

D. H., YIP, C. C., ILES, G. H., AND P. (1972) Cold Spring Harbor Symp.

Quunt. Biol. 37, 469-477. 61. RACKER,

E.,

AND

EYTAN,

Chem. 250. 7533-7534.

E.

(1975)

J. Biol.