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Cyclic AMP-stimulated protein kinase prepared from bovine thyroid glands
Cyclic AMP-stimulated
Protein Kinase Prepared From
Bovine Thyroid
Glands
By KAMEJIRO YAMASHITAAND JAMES B. FIELD A protein kinase that was stimulated threeto fourfold by cyclic AMP (5 X 10-e M) was partially p&tied from bovine thyroid glands. I-&tones and protamine were better substrates for the enzyme than casein or bovine serum albumin, and stimulation induced by cyclic AMP was greater using the former two substances. The concentration of cyclic AMP that gave half-mazimal activation (K,) was 9.1 X lo-8 M, and 5 X 10-e M cyclic AMP produced maximal stimulation. The K, for ATP was 1.3 X 10-S M both in the presence and absence of cyclic AMP. However, cyclic AMP (5 x 1O-6 M) increased the V,,, of the enzyme 3.5fold. Other cyclic nucleotides (cyclic IMP, cyclic GMP, cyclic UMP, cyclic CMP, and dibutyryl-cyclic AMP) also activated the enzyme but only at concentrations considerably higher than that re-
quired for cyclic AMP. Substitution of 2.5 mM Co++ for 2.5 mM Mg+ + increased basal protein khrase activity and the response induced by cyclic AMP. Replacement of Mg++ by Mn++ caused reduction in basal and cyclic AMP mediated activities. Basal enzyme activity was very low when Ca++ was used instead of Mg+ +, and cyclic AMP produced very little stimulation. Adenosine and ADP (5 X 10-b M) markedly inhibited basal and cyclic nucleotide (cyclic AMP, cyclic GMP, and cyclic IMP) activated protein kmase activity. Similar concentrations of FMN, FAD, GDP, adenine, and AMP were either inactive or much less effective. Although these data provide some properties of a cyclic AMP-dependent protein kinase in the thyroid, its role in thyroid gland function still remains to be elucidated.
STIMULATING HORMONE (TSH) seems to regulate thyroid gland function as a consequence of stimulation of the adenyl cyclasecyclic AMP system.1-4 Thus TSH rapidly increases the concentration of cyclic AMP in bovine and canine thyroid slices. 3,4 Most of the effects of TSH on the thyroid have been reproduced by cyclic AMP or its dibutyryl derivative,” but the mechanism by which cyclic AMP mediates these effects has not been elucidated. The recent demonstration of a cyclic AMP-dependent protein kinase in muscle and its ability to activate phosphorylase kinase has provided a possible mechanism for the effects of cyclic AMP. 5 Although this enzyme also phosphorylates histones, protamine, and casein, its physiologic substrate in most tissus is not known. A protein kinase activated by cyclic AMP has been demonstrated in a variety of tissues in which hormones influence tissue cyclic
T
HYROID
From the Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication June 28, 1971. Supported by VSPHS Grant AM-06865 from the National Institutes of Health. KAMEJIRO YAMASHITA, M.D.: Research Associate, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. JAMES B. FIELD, M.D.: Professor of Medicine, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa.
1.50
METABOLISM, VOL. 21, No. 2 (FEBRUARY), 1972
AMP-STIMULATED
151
PROTEIN KINASE
g 0 a
MO-
?
Fig. l.-Effect
2
incubation time on cyclic AMP stimulation of thyroid protein kinase activity. Concentration of cyclic AMP was 5 x 10-e M. of
4cc-
% k 3oog 2 a M e u
/ + C,CllC AMP 7
/
/
IM ,255
20
10 Incubation
Time
(Minutes)
AMP concentrations, and it has been suggested that in these tissues the effects of cyclic AMP reflect activation of this enzyme .5-10In a survey of various tissues, Kuo et al. noted that bovine thyroid contained protein kinase activity that was stimulated by cyclic AMP. lo This paper describes some of the properties of a cyclic AMP responsive protein kinase prepared from bovine thyroid glands. MATERIALS AND METHODS Fresh bovine thyroids, obtained from a local abattoir, were transported to the laboratory on ice. If the tissue was not processed the same day, it was then stored at -2OOC. There did not seem to be any consistent difference between the activity of protein kinase prepared from fresh thyroid and tissue that had been frozen at -2OOC. Protein kinase was partially purified by a modification of the method reported by Miyamoto et al.6 After the DEAEcellulose chromatography step, solid ammonium sulfate (32.5 g/ 100 ml) was added to precipitate the enzyme. The precipitate was collected by centrifugation at 16,000 g for 20 min and dissolved in 5 mM potassium phosphate buffer (pH 7.0) to achieve a volume of 10% of that just prior to the DEAE-cellulose chromatography. After the solution was dialyzed overnight against 50 volumes of the same buffer it was centrifuged at 27,000 g for 30 min, and the supernatant was used for protein kinase assay. All steps were carried out at 4OC. The enzyme preparation was stored in 0.5ml portions at -20°C without any loss of activity for at least 2 mo. The activity of the cyclic AMP-dependent protein kinase was assayed in an incubation volume of 0.2 ml using the procedure of Miyamoto et al.6 The standard assay mixture contained 10 pmoles of sodium glycerol phosphate buffer pH 6.5, 100 fig histone, 32 pmole yJ2P-ATP (300,000 cpm), 1 mJLmole ATP, 2 @moles of MgCl,, 2 floles of NaF, 0.4 sunoles theophylline, and 0.06 &moles of ethylene glycol bis (p-aminoethyl ether)-N, N’-tetracetic acid, with or without 1000 pmoles of cyclic AMP. The reaction was started by the addition of the enzyme (73 Fg protein/30 ~1). The mixture was incubated at 30°C for 10 min in a shaking water bath, and the reaction was terminated by the addition of
F
z
40’)
’
;, 3”1 ” s .- ZLG II Z ,oo Z u
1”2” a
j ,
is Enzyme
IOU- yo.--7 Concentration
bg Protein)
Fig. Z.-Effect of enzyme concentration on protein kinase activity in presence and absence of 5 X 10-s M cyclic AMP.
152
YAMASHITA
AND
FIELD
Fig. 3.-Effect of pH on protein kinase activity in presence and absence of 5 X 10-G M cyclic AMP.
- Cyclrc
AMP
4 ml of 7.5% trichloroacetic
acid solution. Two-tenths milliliter of 0.63% bovine serum albumin was added as carrier protein. The mixture was centrifuged, and the supernatant was removed by aspiration. The precipitate was dissolved in 0.1 ml of 1 N NaOH, and the protein was reprecipitated with 2 ml of 5% trichloroacetic acid. The centrifugation and aspiration steps were repeated twice. The final precipitate was dissolved in 0.1 ml of 1 N NaOH, and 0.5 ml of distilled water was added. The whole solution was added to 5 ml of Triton X-100 and counted in a Packard liquid scintillation spectrometer. A tube containing the complete reaction mixture and boiled enzyme was included in each assay as a blank. Assays were done in triplicate. The enzyme activity was expressed as pmole of P incorporated into protein from y-32P-ATP and ATP/mg protein of enzyme per 10 min at 3O’C. y-s*P-ATP (15.8 Ci/mmole) was purchased from Amersham-Searle Corp. Cyclic AMP and dibutyryl cyclic AMP were obtained from Calbiochemical Corp. Other cyclic nucleotides (cyclic IMP, cyclic GMP, cyclic UMP, and cyclic CMP), ATP, ADP, 5’-AMP, histone (calf thymus), protamine (salmon, histone-free), casein, FMN, and FAD were purchased from Sigma. Bovine serum albumin was obtained from Armour Pharmaceutical co. RESULTS
The data in Fig. 1 demonstrate that protein kinase activity was stimulated by cyclic AMP (5 X 10” M) . Such stimulation was apparent as soon as 1 min, the earliest time tested, and continued to increase for as long as 30 min.
plot of proFig. 4. -Lineweaver-Burke tein kinase activity as function of cyclic AMP concentration.
AMP-STIMULATED
PROTEIN
153
KINASE
Fig. 5.-Effect of ATP concentration on protein kinase activity in presence and absence of 5 X 10-S M cyclic AMP.
ATP
(mM)
Although enzyme activity was linear in the absence of cyclic AMP for the entire 30 min, it was linear for only 10 min when the cyclic nucleotide was added. In the absence of cyclic AMP, protein kinase activity was proportional to the amount of enzyme added up to 122 pg of protein (Fig. 2). In the presence of cyclic AMP, linearity was achieved up to 73 pg of protein. The pH optimum of protein kinase activity was 6.5 both with and without cyclic AMP (Fig. 3 ) . The data in Table 1 indicate that protein kinase activity was increased by 5 X 10-O M cyclic AMP and that maximum stimulation was produced by l-5 X 1O-6 M cyclic AMP. Increasing the cyclic AMP concentration above this actually resulted in less stimulation of protein kinase activity. The K, for cyclic AMP was found to be 9.1 X lo-* M (Fig. 4). The effect of the ATP concentration on protein kinase activity is shown in Fig. 5. LineweaverBurke plot of this data indicates that the K, for ATP was 1.3 X 10e5 M regardless of whether the enzyme was activated by cyclic AMP (Fig. 6). However, in the presence of cyclic AMP (5 X 1O-6 M) the V,,, of the enzyme was increased three and one-half times. Other cyclic nucleotides besides cyclic AMP also stimulated protein kinase activity prepared from bovine thyroid glands (Table 2). Although cyclic AMP was the most effective of the nucleotides tested at 5 X 1O-7 M, at higher concentrations the other cyclic nucleotides caused equivalent or somewhat greater stimulation of protein kinase activity. At 5 X lo-? M cyclic GMP, CMP, UMP, and dibutyryl cyclic AMP had very little effect on the enzyme. Similar to the results obtained with
Fig. 6 .-Lineweaver-Burke presented in Fig. 5.
3 ,’ /’ ,‘___- _-_
I ollm
T co I/ATP
(mM)
/ am
plot of data
154
YAMASHITA Table L-Effect
of Cyclic AMP Concentration
cyclic AMP
Concentration Of)
5 x 1x 5 X 1X 2.5 X 5 X 1.25 x 5 X 1.25 x 5 x 5 x 5 X
FIELD
on Protein Kinase Activity Protein Kinase Activity (pmoles P Incorporated/mg Protein)
0 lo-lO A 10-s M 1O-g M
46 45 48 53
10-B 1O-s
5.5
10-s 10-T IO-7 10-s 10-a 10-s 1O-4
AND
M M M
73 94 126 160 173 182 151 73
M M M M M M
cyclic AMP, stimulation induced by cyclic GMP and IMP was decreased when higher concentrations were used. The data in Table 3 summarize results using various substrates in the protein kinase assay. Although small, but consistent, stimulation of protein kinase by cyclic AMP was observed in the absence of any added substrate, the basal activity was quite low. Histones were a much better substrate both for basal and cyclic AMP stimulated activity. Increasing the amount of histones caused greater basal protein kinase activity, but the effect was not linear. The per cent stimulation induced by cyclic AMP was greatest using 100 pg histones, while larger and smaller amounts gave similar results. Although protamine increased basal protein kinase activity, the stimulation induced by cyclic AMP was not as great as when histones were used. Bovine serum albumin and casein were poor substrates for the enzyme, and the stimulation produced by cyclic AMP was much less than when histones were utilized. The data in Table 4 summarize the effects on protein kinase activity of replacing Mg++by other metal ions. Although increasing the Mg++ concentration to 10 mM augmented basal and cyclic AMP stimulated protein kinase activity, the per cent of the stimulation was unchanged. When equivalent amounts of Mn++ were used instead of Mg++, both basal and cyclic AMP stimulated activities were somewhat reduced. Basal protein kinase activity was increased when Table Z.-Effect Cyclic Nucleotide Concn.
Cyclic AMP Cyclic IMP Cyclic GMP Cyclic UMP Cyclic CMP Dibutyryl Cyclic AMP
of Cyclic Nucleotides 5 x lo-‘M
177 102 55 57 51 50
on Protein Kinase Activity
Protein Kinase Activity ‘p;lo~~,~~I~rporate~/~~~_~o~~)
179 215 138 78 76 77
Protein kinase activity in absence of any cyclic nucleotide mg protein.
175 174 215 156 156 146
5 x 10-L M
82 170 166 190 164 167
was 47 pmoles P incorporated/
AMP-STIMULATEDPROTEINKINASE Table 3.-E&d
155
of Substrates on Protein Kinase Activity Protein Kinase Activity
Substrate
kunoles P Incorporated/mg Protein) - Cyclic AMP Cyclic AMP 5 x 104 M
None Bovine serum albumin Casein
Protamine
Histones
40 100 600 40 100 600 40 100 600 20 40 100 600
6 6 7 7 8 10 11 50 64 75 32 47 65 91
% Stimulation by Cyclic AMP
9
50
8 9 11 11 14 18 95 99 117 66 110 173 191
33 28 56 38 40 63 90 53 57 106 135 167 111
2.5 mM but not 10 miV Co++ was substituted for equivalent amounts of Mg”. The stimulation of enzyme activity produced by cyclic AMP was somewhat greater with Co++ than with Mg++. Ca++ (both 2.5 and 10 mM) markedly inhibited protein kinase activity in the presence and absence of cyclic AMP. Adenosine and ADP (5 X 1O-6 M) reduced basal and cyclic AMP stimulated protein kinase activity by approximately 50% (Table 5). Similar inhibition was apparent when protein kinase activity was stimulated with 5 X 1O-6 M cyclic GMP and IMP. FMN and FAD (5 X low5 M) increased protein kinase activity slightly but did not modify the effect of cyclic AMP. GDP reduced the stimulation caused by cyclic AMP and cyclic GMP (5 X 1O-8 M) but not that due to an equivalent amount of cyclic IMP or a tenfold greater amount of cyclic GMP. Guanosine had no effect on basal or stimulated protein kinase activity. Adenine and AMP slightly inhibited cyclic AMP stimulation of protein kinase activity.
DISCUSSION The characteristics of the protein kmase activity puritied from bovine thyroid tissue indicate that the enzyme is quite similar to that which has been purified from bovine brain.* While these studies were in progress, Kuo et al. described some of the properties of the protein kinase that they purified from bovine Table 4.-Etfects IOIl
Mg+f Mn++ co++ Ca++
of Divalent Cations on Protein Kinase Activity
Protein Khase Activity (pmdes P Incorporates/) -Cyclic AMP cyclic AMP (2.5 m&f) 13 11 31 1
Cyclic AMP concentration
43 29 114 1 was 5 X 10-s M.
30 11 31 0
(10 mM)
cyclic AMP 100 24 133 1
156
YAMASHITA
Table S-Effect
AND
FIELD
of Various Substances on Basal and Cyclic Nucleotide Stimulation of Protein Kinase Activity _____~~~_.__ Protein Kinase Activity (pmoles P Incorporatedjmg Protein)
Substance
None
-Cyclic ____~
Adenosine Guanosine ADP GDP None FMN FAD Adenine AMP __Concentration of ments are shown.
AMP
Cyclic AMP (5 x 10-e M)
54
Cyclic GMP (5 x 10-c M) ____~~ ~~
22 56 27 62 46 61 57 41 63
209 106 182 107 154 192 226 199 165 172
all substances
tested was 5
129 60 122 49 92 X
(5 x 10-j M)
Cyclic IMP (5 x 10” M)
159 85 162 89 145 -
10-S M. Results of two separate
170 93 169 82 186 experi-
thyroid tissue. lo Except for some minor differences, the present results are very similar to those observed by Kuo et al. In their studies the apparent Km for cyclic AMP was 3 X lOA M, while we obtained a value of 9.1 X lo-* M. Both of these values are significantly less than the approximate 1 X 10-F M concentration of cyclic AMP in the thyroid in the absence of TSH.4 This tissue concentration of cyclic AMP is very similar to that which produced maximal stimulation of protein kinase activity and suggests that all of the cyclic AMP that is measured in thyroid tissue may not be available for activation of the enzyme. Under such circumstances the increased amounts of cyclic AMP produced by TSH would still augment protein kinase activity and mediate those metabolic effects of TSH that reflect stimulation of adenyl cyclase. Increasing the cyclic AMP concentration to 5 X lo-” and 5 X 1O-4 M caused decreased activation of protein kinase similar to what has been reported for the enzyme prepared from other tissues.8J0 Whether the enzyme was stimulated by cyclic AMP or not, K,,, for ATP was 1.3 X lo-‘, M. Although this value was very similar to that of cyclic AMPstimulated protein kinase purified from bovine brain, Miyamoto et al. reported that the apparent K, for ATP in the absence of cyclic AMP was 2.2 X lo-” M for the brain enzyme.s This same laboratory also reported that in the absence of cyclic AMP greater concentrations of ATP were necessary to achieve halfmaximal velocities utilizing protein kinase prepared from pancreas, kidney, heart, and lung.lO In our experiments cyclic AMP (5 X 10-O M) caused a 3.5-fold increase in the V,,, of the protein kinase prepared from thyroid. A similar increase in V,,, induced by cyclic AMP was reported using brain protein kinase.8 The divalent cation requirements of protein kinases isolated from a variety of different organs have been very similar.lO In contrast to the results obtained by Kuo et al.l” using thyroid tissue, we found that replacement of Mg” with Mn++ decreased protein kinase activity, while substitution of CO++ for Mg++ augmented both basal and cyclic AMP-stimulated enzyme
AMP-STIMULATED
157
PROTEIN KINASE
activity. Our results confirmed theirs in regard to the inhibitory effects of Ca++on both basal and cyclic AMP stimulation of protein kinase activity. Histones and protamine were equivalent as substrates for basal activity of protein kinase isolated from bovine thyroid, although in the presence of 5 X 1O-8 M cyclic AMP histones were better substrates. Equivalent amounts of casein and bovine serum albumin were poor substrates for the enzyme both in the presence and absence of cyclic AMP. The very low activity in the absence of added substrate and the small increase induced by cyclic AMP provide no clues as to the physiologic substrate for the enzyme in thyroid tissue. It has been speculated that the phosphorylation of histones might modify DNA-dependent RNA synthesis and thus mediate hormonal effects that might be related to increased protein synthesis. 6 However, such action would not explain the TSH stimulation of 14C-l-glucose oxidation or 32P incorporation into phospholipids, since neither of these effects is inhibited by puromycin.11J2 The specificity of the cyclic nucleotide stimulation of thyroid protein kinase is very similar to that which has been reported for the enzyme prepared from other organslo The enzyme was most sensitive to cyclic AMP, but equivalent stimulation could be obtained when higher concentrations of cyclic IMP, GMP, UMP, CMP, and dibutyryl cyclic AMP were used. The significance of the effects of these other nucleotides is questionable, since they are much less effective than cyclic AMP at lower concentrations. Although Pisarev et al. reported that cyclic GMP, similar to cyclic AMP,13 increased 3H-leucine incorporation into protein, total thyroid protein, and thyroid weight in thyroxine suppressed mice,14 we have been unable to measure an increase in cyclic GMP in thyroid slices incubated with TSH (unpublished observations). Our results utilizing various derivatives of adenine generally agree quite well with those reported by Kuo et al.,l” except that we did not find any inhibitory effect of FMN. Adenosine and ADP were very similar in their inhibitory action. These latter two compounds also inhibited the cyclic GMP and IMP stimulation of protein kinase activity. The analogous guanine derivatives, however, had no consistent effect on the stimulation caused by cyclic AMP, GMP or IMP. The demonstration of a cyclic nucleotide-stimulated protein kinase in thyroid tissue is compatible with the current concept that many, if not all, of the effects of cyclic AMP in tissues can be attributed to the activation of protein kinase.lO Recent work has clarified the mechanism by which cyclic AMP activates protein kinase activity and has defined two components of the system.15 The binding of cyclic AMP to the regulatory unit appears to dissociate it from the catalytic unit and results in greater enzyme activity. Although this mechanism may be common to a large number of tissues, the specificity of hormonal effects would reflect the proteins that would be phosphorylated by the protein kinases. The physiologic substrates for such phosphorylation in the thyroid remain to be delined, as well as the mechanism by which such changes produce the multiple effects resulting from TSH action. REFERENCES 1. Pastan, I., and Katzen, R.: Activation of adenyl cyclase in thyroid homogenates by
thyroid-stimulating hormone. Biochem. phys. Res. Commun. 29:792, 1967.
Bio-
158 2. Zor, U., Kaneko, T., Lowe, I. P., Bloom, G., and Field, J. B.: Effect of thyroid-stimulating hormone and prostaglandins on thyroid adenyl cyclase activation and cyclic adenosine 3’,.5’-monophosphate. J. Biol. Chem. 244:5189, 1969. 3. Gilman, A. G., and Rall, T. W.: Factors influencing adenosine 3’,5’-phosphate accumulation in bovine thyroid slices. J. Biol. Chem. 243:5867, 1968. 4. Kaneko, T., Zor, U., and Field, J. B.: Thyroid-stimulating hormone and prostaglandin E, stimulation of cyclic 3’,5’adenosine monophosphate in thyroid slices. Science 163: 1062, 1969. 5. Walsh, D. A., Perkins, J. P., and Krebs, E. G.: An adenosine 3’5’-monophosphate-dependent protein kinase from rabbit skeletal muscle. J. Biol. Chem. 243: 3763, 1968. 6. Langan, T. A.: Histone phosphorylation: Stimulation by adenosine3’,5’-monophosphate. Science 162:579, 1968. 7. Corbin, J. D., and Krebs, E. G.: A cyclic AMP-stimulated protein kinase in adipose tissue. Biochem. Biophys. Res. Commun. 36:328, 1969. 8. Miyamoto, E., Kuo, J. F., and Greengard, P.: Cyclic nucleotide-dependent protein kinases III. Purification and properties of adenosine 3’,5’-monophosphate-dependent protein kinase from bovine brain. J. Biol. Chem. 244:6395, 1969. 9. Kuo, J. F., and Greengard, P.: Cyclic nucleotide-dependent protein kinases. IV. Widespread occurrence of adenosine 3’,5’-
YAMASHITA
AND FIELD
monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom. Proc. Nat. Acad. Sci. USA 64: 1349, 1969. 10. -, Krueger, B. K., Sane, J. R., and Greengard, P.: Cyclic nucleotide-dependent protein kinases V. Preparation and propcrties of adenosine 3’,5’-monophosphate-dependent protein kinase from various bovine tissues. Biochim. Biophys. Acta 212:79, 1970. 11. Field, J. B., Johnson, P., Kendig, E., and Pastan, I.: Further studies on effects of thyroid-stimulating hormone on thyroid glucose oxidation. J. Biol. Chem. 238:1189, 1963. 12. Oka, H., and Field, J. B.: Effects of ions and ouabain on thyroid stimulating hormone of Psa incorporation into phospholipid in dog thyroid slices. Amer. J. Physiol. 211:1357, 1966. 13. Pisarev, M. A., DeGroot, L. I., and Wilber, J. F.: Cyclic-AMP production of goiter. Endocrinology 87: 339, 1970. 14. -, -, -, and Altschuler, N.: Action of cyclic guanosine monophosphate on thyroid weight and protein. Endocrinology 88: 1074, 1971. 15. Reimann, E. M., Brostrom, C. O., Corbin, J. D., King, C. A., and Krebs, E. G.: Separation of regulatory and catalytic subunits of the cyclic 3’,5’-adenosine monophosphate-dependent protein kinase(s) of rabbit skeletal muscle. Biochem. Biophys. Res. Commun. 42:187, 1971.
Protein Kinase Prepared From
Bovine Thyroid
Glands
By KAMEJIRO YAMASHITAAND JAMES B. FIELD A protein kinase that was stimulated threeto fourfold by cyclic AMP (5 X 10-e M) was partially p&tied from bovine thyroid glands. I-&tones and protamine were better substrates for the enzyme than casein or bovine serum albumin, and stimulation induced by cyclic AMP was greater using the former two substances. The concentration of cyclic AMP that gave half-mazimal activation (K,) was 9.1 X lo-8 M, and 5 X 10-e M cyclic AMP produced maximal stimulation. The K, for ATP was 1.3 X 10-S M both in the presence and absence of cyclic AMP. However, cyclic AMP (5 x 1O-6 M) increased the V,,, of the enzyme 3.5fold. Other cyclic nucleotides (cyclic IMP, cyclic GMP, cyclic UMP, cyclic CMP, and dibutyryl-cyclic AMP) also activated the enzyme but only at concentrations considerably higher than that re-
quired for cyclic AMP. Substitution of 2.5 mM Co++ for 2.5 mM Mg+ + increased basal protein khrase activity and the response induced by cyclic AMP. Replacement of Mg++ by Mn++ caused reduction in basal and cyclic AMP mediated activities. Basal enzyme activity was very low when Ca++ was used instead of Mg+ +, and cyclic AMP produced very little stimulation. Adenosine and ADP (5 X 10-b M) markedly inhibited basal and cyclic nucleotide (cyclic AMP, cyclic GMP, and cyclic IMP) activated protein kmase activity. Similar concentrations of FMN, FAD, GDP, adenine, and AMP were either inactive or much less effective. Although these data provide some properties of a cyclic AMP-dependent protein kinase in the thyroid, its role in thyroid gland function still remains to be elucidated.
STIMULATING HORMONE (TSH) seems to regulate thyroid gland function as a consequence of stimulation of the adenyl cyclasecyclic AMP system.1-4 Thus TSH rapidly increases the concentration of cyclic AMP in bovine and canine thyroid slices. 3,4 Most of the effects of TSH on the thyroid have been reproduced by cyclic AMP or its dibutyryl derivative,” but the mechanism by which cyclic AMP mediates these effects has not been elucidated. The recent demonstration of a cyclic AMP-dependent protein kinase in muscle and its ability to activate phosphorylase kinase has provided a possible mechanism for the effects of cyclic AMP. 5 Although this enzyme also phosphorylates histones, protamine, and casein, its physiologic substrate in most tissus is not known. A protein kinase activated by cyclic AMP has been demonstrated in a variety of tissues in which hormones influence tissue cyclic
T
HYROID
From the Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication June 28, 1971. Supported by VSPHS Grant AM-06865 from the National Institutes of Health. KAMEJIRO YAMASHITA, M.D.: Research Associate, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. JAMES B. FIELD, M.D.: Professor of Medicine, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa.
1.50
METABOLISM, VOL. 21, No. 2 (FEBRUARY), 1972
AMP-STIMULATED
151
PROTEIN KINASE
g 0 a
MO-
?
Fig. l.-Effect
2
incubation time on cyclic AMP stimulation of thyroid protein kinase activity. Concentration of cyclic AMP was 5 x 10-e M. of
4cc-
% k 3oog 2 a M e u
/ + C,CllC AMP 7
/
/
IM ,255
20
10 Incubation
Time
(Minutes)
AMP concentrations, and it has been suggested that in these tissues the effects of cyclic AMP reflect activation of this enzyme .5-10In a survey of various tissues, Kuo et al. noted that bovine thyroid contained protein kinase activity that was stimulated by cyclic AMP. lo This paper describes some of the properties of a cyclic AMP responsive protein kinase prepared from bovine thyroid glands. MATERIALS AND METHODS Fresh bovine thyroids, obtained from a local abattoir, were transported to the laboratory on ice. If the tissue was not processed the same day, it was then stored at -2OOC. There did not seem to be any consistent difference between the activity of protein kinase prepared from fresh thyroid and tissue that had been frozen at -2OOC. Protein kinase was partially purified by a modification of the method reported by Miyamoto et al.6 After the DEAEcellulose chromatography step, solid ammonium sulfate (32.5 g/ 100 ml) was added to precipitate the enzyme. The precipitate was collected by centrifugation at 16,000 g for 20 min and dissolved in 5 mM potassium phosphate buffer (pH 7.0) to achieve a volume of 10% of that just prior to the DEAE-cellulose chromatography. After the solution was dialyzed overnight against 50 volumes of the same buffer it was centrifuged at 27,000 g for 30 min, and the supernatant was used for protein kinase assay. All steps were carried out at 4OC. The enzyme preparation was stored in 0.5ml portions at -20°C without any loss of activity for at least 2 mo. The activity of the cyclic AMP-dependent protein kinase was assayed in an incubation volume of 0.2 ml using the procedure of Miyamoto et al.6 The standard assay mixture contained 10 pmoles of sodium glycerol phosphate buffer pH 6.5, 100 fig histone, 32 pmole yJ2P-ATP (300,000 cpm), 1 mJLmole ATP, 2 @moles of MgCl,, 2 floles of NaF, 0.4 sunoles theophylline, and 0.06 &moles of ethylene glycol bis (p-aminoethyl ether)-N, N’-tetracetic acid, with or without 1000 pmoles of cyclic AMP. The reaction was started by the addition of the enzyme (73 Fg protein/30 ~1). The mixture was incubated at 30°C for 10 min in a shaking water bath, and the reaction was terminated by the addition of
F
z
40’)
’
;, 3”1 ” s .- ZLG II Z ,oo Z u
1”2” a
j ,
is Enzyme
IOU- yo.--7 Concentration
bg Protein)
Fig. Z.-Effect of enzyme concentration on protein kinase activity in presence and absence of 5 X 10-s M cyclic AMP.
152
YAMASHITA
AND
FIELD
Fig. 3.-Effect of pH on protein kinase activity in presence and absence of 5 X 10-G M cyclic AMP.
- Cyclrc
AMP
4 ml of 7.5% trichloroacetic
acid solution. Two-tenths milliliter of 0.63% bovine serum albumin was added as carrier protein. The mixture was centrifuged, and the supernatant was removed by aspiration. The precipitate was dissolved in 0.1 ml of 1 N NaOH, and the protein was reprecipitated with 2 ml of 5% trichloroacetic acid. The centrifugation and aspiration steps were repeated twice. The final precipitate was dissolved in 0.1 ml of 1 N NaOH, and 0.5 ml of distilled water was added. The whole solution was added to 5 ml of Triton X-100 and counted in a Packard liquid scintillation spectrometer. A tube containing the complete reaction mixture and boiled enzyme was included in each assay as a blank. Assays were done in triplicate. The enzyme activity was expressed as pmole of P incorporated into protein from y-32P-ATP and ATP/mg protein of enzyme per 10 min at 3O’C. y-s*P-ATP (15.8 Ci/mmole) was purchased from Amersham-Searle Corp. Cyclic AMP and dibutyryl cyclic AMP were obtained from Calbiochemical Corp. Other cyclic nucleotides (cyclic IMP, cyclic GMP, cyclic UMP, and cyclic CMP), ATP, ADP, 5’-AMP, histone (calf thymus), protamine (salmon, histone-free), casein, FMN, and FAD were purchased from Sigma. Bovine serum albumin was obtained from Armour Pharmaceutical co. RESULTS
The data in Fig. 1 demonstrate that protein kinase activity was stimulated by cyclic AMP (5 X 10” M) . Such stimulation was apparent as soon as 1 min, the earliest time tested, and continued to increase for as long as 30 min.
plot of proFig. 4. -Lineweaver-Burke tein kinase activity as function of cyclic AMP concentration.
AMP-STIMULATED
PROTEIN
153
KINASE
Fig. 5.-Effect of ATP concentration on protein kinase activity in presence and absence of 5 X 10-S M cyclic AMP.
ATP
(mM)
Although enzyme activity was linear in the absence of cyclic AMP for the entire 30 min, it was linear for only 10 min when the cyclic nucleotide was added. In the absence of cyclic AMP, protein kinase activity was proportional to the amount of enzyme added up to 122 pg of protein (Fig. 2). In the presence of cyclic AMP, linearity was achieved up to 73 pg of protein. The pH optimum of protein kinase activity was 6.5 both with and without cyclic AMP (Fig. 3 ) . The data in Table 1 indicate that protein kinase activity was increased by 5 X 10-O M cyclic AMP and that maximum stimulation was produced by l-5 X 1O-6 M cyclic AMP. Increasing the cyclic AMP concentration above this actually resulted in less stimulation of protein kinase activity. The K, for cyclic AMP was found to be 9.1 X lo-* M (Fig. 4). The effect of the ATP concentration on protein kinase activity is shown in Fig. 5. LineweaverBurke plot of this data indicates that the K, for ATP was 1.3 X 10e5 M regardless of whether the enzyme was activated by cyclic AMP (Fig. 6). However, in the presence of cyclic AMP (5 X 1O-6 M) the V,,, of the enzyme was increased three and one-half times. Other cyclic nucleotides besides cyclic AMP also stimulated protein kinase activity prepared from bovine thyroid glands (Table 2). Although cyclic AMP was the most effective of the nucleotides tested at 5 X 1O-7 M, at higher concentrations the other cyclic nucleotides caused equivalent or somewhat greater stimulation of protein kinase activity. At 5 X lo-? M cyclic GMP, CMP, UMP, and dibutyryl cyclic AMP had very little effect on the enzyme. Similar to the results obtained with
Fig. 6 .-Lineweaver-Burke presented in Fig. 5.
3 ,’ /’ ,‘___- _-_
I ollm
T co I/ATP
(mM)
/ am
plot of data
154
YAMASHITA Table L-Effect
of Cyclic AMP Concentration
cyclic AMP
Concentration Of)
5 x 1x 5 X 1X 2.5 X 5 X 1.25 x 5 X 1.25 x 5 x 5 x 5 X
FIELD
on Protein Kinase Activity Protein Kinase Activity (pmoles P Incorporated/mg Protein)
0 lo-lO A 10-s M 1O-g M
46 45 48 53
10-B 1O-s
5.5
10-s 10-T IO-7 10-s 10-a 10-s 1O-4
AND
M M M
73 94 126 160 173 182 151 73
M M M M M M
cyclic AMP, stimulation induced by cyclic GMP and IMP was decreased when higher concentrations were used. The data in Table 3 summarize results using various substrates in the protein kinase assay. Although small, but consistent, stimulation of protein kinase by cyclic AMP was observed in the absence of any added substrate, the basal activity was quite low. Histones were a much better substrate both for basal and cyclic AMP stimulated activity. Increasing the amount of histones caused greater basal protein kinase activity, but the effect was not linear. The per cent stimulation induced by cyclic AMP was greatest using 100 pg histones, while larger and smaller amounts gave similar results. Although protamine increased basal protein kinase activity, the stimulation induced by cyclic AMP was not as great as when histones were used. Bovine serum albumin and casein were poor substrates for the enzyme, and the stimulation produced by cyclic AMP was much less than when histones were utilized. The data in Table 4 summarize the effects on protein kinase activity of replacing Mg++by other metal ions. Although increasing the Mg++ concentration to 10 mM augmented basal and cyclic AMP stimulated protein kinase activity, the per cent of the stimulation was unchanged. When equivalent amounts of Mn++ were used instead of Mg++, both basal and cyclic AMP stimulated activities were somewhat reduced. Basal protein kinase activity was increased when Table Z.-Effect Cyclic Nucleotide Concn.
Cyclic AMP Cyclic IMP Cyclic GMP Cyclic UMP Cyclic CMP Dibutyryl Cyclic AMP
of Cyclic Nucleotides 5 x lo-‘M
177 102 55 57 51 50
on Protein Kinase Activity
Protein Kinase Activity ‘p;lo~~,~~I~rporate~/~~~_~o~~)
179 215 138 78 76 77
Protein kinase activity in absence of any cyclic nucleotide mg protein.
175 174 215 156 156 146
5 x 10-L M
82 170 166 190 164 167
was 47 pmoles P incorporated/
AMP-STIMULATEDPROTEINKINASE Table 3.-E&d
155
of Substrates on Protein Kinase Activity Protein Kinase Activity
Substrate
kunoles P Incorporated/mg Protein) - Cyclic AMP Cyclic AMP 5 x 104 M
None Bovine serum albumin Casein
Protamine
Histones
40 100 600 40 100 600 40 100 600 20 40 100 600
6 6 7 7 8 10 11 50 64 75 32 47 65 91
% Stimulation by Cyclic AMP
9
50
8 9 11 11 14 18 95 99 117 66 110 173 191
33 28 56 38 40 63 90 53 57 106 135 167 111
2.5 mM but not 10 miV Co++ was substituted for equivalent amounts of Mg”. The stimulation of enzyme activity produced by cyclic AMP was somewhat greater with Co++ than with Mg++. Ca++ (both 2.5 and 10 mM) markedly inhibited protein kinase activity in the presence and absence of cyclic AMP. Adenosine and ADP (5 X 1O-6 M) reduced basal and cyclic AMP stimulated protein kinase activity by approximately 50% (Table 5). Similar inhibition was apparent when protein kinase activity was stimulated with 5 X 1O-6 M cyclic GMP and IMP. FMN and FAD (5 X low5 M) increased protein kinase activity slightly but did not modify the effect of cyclic AMP. GDP reduced the stimulation caused by cyclic AMP and cyclic GMP (5 X 1O-8 M) but not that due to an equivalent amount of cyclic IMP or a tenfold greater amount of cyclic GMP. Guanosine had no effect on basal or stimulated protein kinase activity. Adenine and AMP slightly inhibited cyclic AMP stimulation of protein kinase activity.
DISCUSSION The characteristics of the protein kmase activity puritied from bovine thyroid tissue indicate that the enzyme is quite similar to that which has been purified from bovine brain.* While these studies were in progress, Kuo et al. described some of the properties of the protein kinase that they purified from bovine Table 4.-Etfects IOIl
Mg+f Mn++ co++ Ca++
of Divalent Cations on Protein Kinase Activity
Protein Khase Activity (pmdes P Incorporates/) -Cyclic AMP cyclic AMP (2.5 m&f) 13 11 31 1
Cyclic AMP concentration
43 29 114 1 was 5 X 10-s M.
30 11 31 0
(10 mM)
cyclic AMP 100 24 133 1
156
YAMASHITA
Table S-Effect
AND
FIELD
of Various Substances on Basal and Cyclic Nucleotide Stimulation of Protein Kinase Activity _____~~~_.__ Protein Kinase Activity (pmoles P Incorporatedjmg Protein)
Substance
None
-Cyclic ____~
Adenosine Guanosine ADP GDP None FMN FAD Adenine AMP __Concentration of ments are shown.
AMP
Cyclic AMP (5 x 10-e M)
54
Cyclic GMP (5 x 10-c M) ____~~ ~~
22 56 27 62 46 61 57 41 63
209 106 182 107 154 192 226 199 165 172
all substances
tested was 5
129 60 122 49 92 X
(5 x 10-j M)
Cyclic IMP (5 x 10” M)
159 85 162 89 145 -
10-S M. Results of two separate
170 93 169 82 186 experi-
thyroid tissue. lo Except for some minor differences, the present results are very similar to those observed by Kuo et al. In their studies the apparent Km for cyclic AMP was 3 X lOA M, while we obtained a value of 9.1 X lo-* M. Both of these values are significantly less than the approximate 1 X 10-F M concentration of cyclic AMP in the thyroid in the absence of TSH.4 This tissue concentration of cyclic AMP is very similar to that which produced maximal stimulation of protein kinase activity and suggests that all of the cyclic AMP that is measured in thyroid tissue may not be available for activation of the enzyme. Under such circumstances the increased amounts of cyclic AMP produced by TSH would still augment protein kinase activity and mediate those metabolic effects of TSH that reflect stimulation of adenyl cyclase. Increasing the cyclic AMP concentration to 5 X lo-” and 5 X 1O-4 M caused decreased activation of protein kinase similar to what has been reported for the enzyme prepared from other tissues.8J0 Whether the enzyme was stimulated by cyclic AMP or not, K,,, for ATP was 1.3 X lo-‘, M. Although this value was very similar to that of cyclic AMPstimulated protein kinase purified from bovine brain, Miyamoto et al. reported that the apparent K, for ATP in the absence of cyclic AMP was 2.2 X lo-” M for the brain enzyme.s This same laboratory also reported that in the absence of cyclic AMP greater concentrations of ATP were necessary to achieve halfmaximal velocities utilizing protein kinase prepared from pancreas, kidney, heart, and lung.lO In our experiments cyclic AMP (5 X 10-O M) caused a 3.5-fold increase in the V,,, of the protein kinase prepared from thyroid. A similar increase in V,,, induced by cyclic AMP was reported using brain protein kinase.8 The divalent cation requirements of protein kinases isolated from a variety of different organs have been very similar.lO In contrast to the results obtained by Kuo et al.l” using thyroid tissue, we found that replacement of Mg” with Mn++ decreased protein kinase activity, while substitution of CO++ for Mg++ augmented both basal and cyclic AMP-stimulated enzyme
AMP-STIMULATED
157
PROTEIN KINASE
activity. Our results confirmed theirs in regard to the inhibitory effects of Ca++on both basal and cyclic AMP stimulation of protein kinase activity. Histones and protamine were equivalent as substrates for basal activity of protein kinase isolated from bovine thyroid, although in the presence of 5 X 1O-8 M cyclic AMP histones were better substrates. Equivalent amounts of casein and bovine serum albumin were poor substrates for the enzyme both in the presence and absence of cyclic AMP. The very low activity in the absence of added substrate and the small increase induced by cyclic AMP provide no clues as to the physiologic substrate for the enzyme in thyroid tissue. It has been speculated that the phosphorylation of histones might modify DNA-dependent RNA synthesis and thus mediate hormonal effects that might be related to increased protein synthesis. 6 However, such action would not explain the TSH stimulation of 14C-l-glucose oxidation or 32P incorporation into phospholipids, since neither of these effects is inhibited by puromycin.11J2 The specificity of the cyclic nucleotide stimulation of thyroid protein kinase is very similar to that which has been reported for the enzyme prepared from other organslo The enzyme was most sensitive to cyclic AMP, but equivalent stimulation could be obtained when higher concentrations of cyclic IMP, GMP, UMP, CMP, and dibutyryl cyclic AMP were used. The significance of the effects of these other nucleotides is questionable, since they are much less effective than cyclic AMP at lower concentrations. Although Pisarev et al. reported that cyclic GMP, similar to cyclic AMP,13 increased 3H-leucine incorporation into protein, total thyroid protein, and thyroid weight in thyroxine suppressed mice,14 we have been unable to measure an increase in cyclic GMP in thyroid slices incubated with TSH (unpublished observations). Our results utilizing various derivatives of adenine generally agree quite well with those reported by Kuo et al.,l” except that we did not find any inhibitory effect of FMN. Adenosine and ADP were very similar in their inhibitory action. These latter two compounds also inhibited the cyclic GMP and IMP stimulation of protein kinase activity. The analogous guanine derivatives, however, had no consistent effect on the stimulation caused by cyclic AMP, GMP or IMP. The demonstration of a cyclic nucleotide-stimulated protein kinase in thyroid tissue is compatible with the current concept that many, if not all, of the effects of cyclic AMP in tissues can be attributed to the activation of protein kinase.lO Recent work has clarified the mechanism by which cyclic AMP activates protein kinase activity and has defined two components of the system.15 The binding of cyclic AMP to the regulatory unit appears to dissociate it from the catalytic unit and results in greater enzyme activity. Although this mechanism may be common to a large number of tissues, the specificity of hormonal effects would reflect the proteins that would be phosphorylated by the protein kinases. The physiologic substrates for such phosphorylation in the thyroid remain to be delined, as well as the mechanism by which such changes produce the multiple effects resulting from TSH action. REFERENCES 1. Pastan, I., and Katzen, R.: Activation of adenyl cyclase in thyroid homogenates by
thyroid-stimulating hormone. Biochem. phys. Res. Commun. 29:792, 1967.
Bio-
158 2. Zor, U., Kaneko, T., Lowe, I. P., Bloom, G., and Field, J. B.: Effect of thyroid-stimulating hormone and prostaglandins on thyroid adenyl cyclase activation and cyclic adenosine 3’,.5’-monophosphate. J. Biol. Chem. 244:5189, 1969. 3. Gilman, A. G., and Rall, T. W.: Factors influencing adenosine 3’,5’-phosphate accumulation in bovine thyroid slices. J. Biol. Chem. 243:5867, 1968. 4. Kaneko, T., Zor, U., and Field, J. B.: Thyroid-stimulating hormone and prostaglandin E, stimulation of cyclic 3’,5’adenosine monophosphate in thyroid slices. Science 163: 1062, 1969. 5. Walsh, D. A., Perkins, J. P., and Krebs, E. G.: An adenosine 3’5’-monophosphate-dependent protein kinase from rabbit skeletal muscle. J. Biol. Chem. 243: 3763, 1968. 6. Langan, T. A.: Histone phosphorylation: Stimulation by adenosine3’,5’-monophosphate. Science 162:579, 1968. 7. Corbin, J. D., and Krebs, E. G.: A cyclic AMP-stimulated protein kinase in adipose tissue. Biochem. Biophys. Res. Commun. 36:328, 1969. 8. Miyamoto, E., Kuo, J. F., and Greengard, P.: Cyclic nucleotide-dependent protein kinases III. Purification and properties of adenosine 3’,5’-monophosphate-dependent protein kinase from bovine brain. J. Biol. Chem. 244:6395, 1969. 9. Kuo, J. F., and Greengard, P.: Cyclic nucleotide-dependent protein kinases. IV. Widespread occurrence of adenosine 3’,5’-
YAMASHITA
AND FIELD
monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom. Proc. Nat. Acad. Sci. USA 64: 1349, 1969. 10. -, Krueger, B. K., Sane, J. R., and Greengard, P.: Cyclic nucleotide-dependent protein kinases V. Preparation and propcrties of adenosine 3’,5’-monophosphate-dependent protein kinase from various bovine tissues. Biochim. Biophys. Acta 212:79, 1970. 11. Field, J. B., Johnson, P., Kendig, E., and Pastan, I.: Further studies on effects of thyroid-stimulating hormone on thyroid glucose oxidation. J. Biol. Chem. 238:1189, 1963. 12. Oka, H., and Field, J. B.: Effects of ions and ouabain on thyroid stimulating hormone of Psa incorporation into phospholipid in dog thyroid slices. Amer. J. Physiol. 211:1357, 1966. 13. Pisarev, M. A., DeGroot, L. I., and Wilber, J. F.: Cyclic-AMP production of goiter. Endocrinology 87: 339, 1970. 14. -, -, -, and Altschuler, N.: Action of cyclic guanosine monophosphate on thyroid weight and protein. Endocrinology 88: 1074, 1971. 15. Reimann, E. M., Brostrom, C. O., Corbin, J. D., King, C. A., and Krebs, E. G.: Separation of regulatory and catalytic subunits of the cyclic 3’,5’-adenosine monophosphate-dependent protein kinase(s) of rabbit skeletal muscle. Biochem. Biophys. Res. Commun. 42:187, 1971.