OF BIOCHEMISTRY AND BIOPHYSICS Vol. 197, No. 1, October 1, pp. 185-192, 1979
ARCHIVES
Ontogeny MAIJA
and Multiplicity of Cyclic AMP-Dependent in Thymic Lymphoid Cells1
I. MEDNIEKS,
Department
RICHARD
of Biochemistry,
Northwestern
A. JUNGMANN, University
AND
Medical
Protein
JOHN
School,
Chicago,
Kinase
S. SCHWEPPE Illinois
60611
Received January 23, 1979; revised April 12, 1979 Cyclic adenosine 3’:5’-monophosphate-dependent protein kinases were studied in thymus lymphoid cells and were found to be similar to their counterparts in other tissues with respect to substrate preference and concentration dependence. A previously not identified, restrictive subcellular compartmentalization of the protein kinase isozymes was found: Type I was predominantly present in the nucleus of adult and juvenile human and rat thymus cells, whereas the type II kinase was restricted to the cytosol fraction of unstimulated cells. Additionally, a decline in the specific activity of protein kinase was progressive with increasing age of the animal and distinct from the general observation that lymphoid cell numbers decrease with age. These findings may be correlated with age-dependent immunodeficiencies and perhaps have functional significance in the regulatory role of protein phosphorylation in lymphoid cell activation.
Lymphoid cells, as do virtually all mammalian cells, contain cyclic adenosine 3’:5’-monophosphate (cyclic AMP) and respond to regulation by a number of environmental stimuli with changes in cyclic nucleotide levels (1). Physiological events which are mediated by cyclic nucleotides in eucaryotic cells involve phosphorylation of protein substrates (2-4) by cyclic AMP-dependent protein kinase (EC 2.7.1.37). Using immunocytochemical techniques (5, 6) distinct intracellular cyclic AMP pools have been identified in lymphocytes, and nuclear protein phosphorylation has been shown to be related to changes in cyclic AMP pools in these cells (7, 8). Information regarding the biochemical characteristics of cyclic AMP-dependent protein kinase in lymphoid cells is therefore relevant to understanding nuclear events which participate in the regulation of immune responses. The present study examines biochemical characteristics of both nuclear and cytoplasmic cyclic AMP-dependent protein kinases and some aspects of its ontogeny 1 This study was in part supported by the Research and Education Fund, Northwestern Memorial Hospital.
in a selected population of lymphoid cells. Thymic tissue has been used in similar studies (9, 10) and provides a convenient source of cells which are relatively free of contaminants in the form of other cell types such as red and white blood cells or fat and connective tissue components. Cell homogeneity becomes a critical consideration when the presence and distribution of protein kinase isozymes is measured. Also, since some biochemical data are available from normal and transformed T-cells (11, 12), comparisons are feasible regarding the intracellular distribution and activity of soluble and particulate protein kinases. It is apparent from our studies that cyclic AMP-dependent protein kinases exhibit a biochemical constancy in thymic cells from humans and from various animals. This finding provides a suitable basis for studying molecular mechanisms of lymphoid cell stimulation in an animal model and might be applied to examining analogous normal and neoplastic human lymphoid cell systems. MATERIALS Tissue
Fresh 185
preparation
thymus
AND METHODS and
tissue
subcellular
fractionation.
was placed
in phosphate
0003-9861/79/110185-O&$02.00/0
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction
in any form reserved.
186
MEDNIEKS.
JUNGMANN.
buffered saline (PBS),’ pH 7.5 (GIBCO, Grand Island, N. Y.) and kept on ice until use. Connective tissue, visible blood vessels, fat, and parathymic lymph nodes were stripped away and thymus cells were separated by gently pressing the tissue through a number 80 mesh (60 pm) stainless-steel screen (Fisher Scientific Co.) with a rubber tipped plunger of a ~-CC syringe. The cells were pelleted using a desk-top clinical centrifuge, resuspended in PBS, strained through gauze, and collected by centrifugation. The washed cells were homogenized in 0.25 M sucrose, 2 mM CaCl,, 4 mM theophylline, 50 mM Tris, pH 7.4 buffer. A crude nuclear pellet was obtained by centrifugation for 20 min at 9OOg. Purified nuclei were obtained by subsequent centrifugation at 105,OOOg for 1 h through 2.0 M sucrose, containing 2 mM CaCI,, 50 mM Tris, pH 7.4. The purified nuclear pellet was rinsed with PBS and extracted with a 0.35 M NaCl, 50 mM MgCI,, 3 mM dithiothreitol, 20 mM Tris, pH 7.4 solution. The nuclear extract and the cytosol (obtained by centrifuging the 900s supernatant at 105,OOOg for 1 h) were then dialyzed against two changes of 5 mM MgCl,, 5 mM mercaptoethanol, 5% glycerol, in 20 mM Tris, pH 7.4. The dialyzed solutions were cleared of precipitated material by centrifugation and subsequently used for enzyme assays and DEAE-Sepharose (Pharmacia) chromatography. Determination of protein kinase activity. The assay was carried out as described previously (13) in a volume of 0.25 ml containing 50 p,g of protamine sulfate or histone Hl, 5 mM a-glycerol phosphate buffer, pH 7.5, 10 mM [y-3ZP]ATP (0.5 &i) without and with cyclic AMP (10m6M in assaying cell fractions and lo-’ M in assaying column fractions), as indicated. Incubation was carried out for 10 min at 30°C and the reaction was stopped by adding 2 ml of 15% trichloroacetic acid (TCA) containing 1% sodium dodecyl sulfate (SDS). The samples were filtered using Millipore filters and washed with the TCA-SDS solution. Radioactivity was measured in a Packard Tri-Carb liquid scintillation counter (Model 3375). Assay reagents were purchased from Sigma Chemical Company. Protein content was determined by the Lowry method (14). Protein kinase activity was measured in cell fractions containing between 20 and 50 fig protein per assay, a range in which phosphate incorporation was linear for all tissues tested. Under these experimental conditions incorporation of [“‘PIphosphate into substrate protein was linear over a 20-min incubation period. Preparation of inhibitor and of antiserum to protein kinase. A heat-stable protein kinase inhibitor preparation was obtained according to the method of * Abbreviations used: PBS, Phosphate buffered saline; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate.
AND SCHWEPPE Walsh et al. (15) from rabbit skeletal muscle. The inhibitor was purified on DEAE-cellulose and used in 5 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA at a concentration of 1 mg of proteinlmi. This preparation was used in conjunction with immunochemical methods to establish the extent of cyclic AMP-independent phosphorylation in our preparations. Antiserum to cyclic AMP-dependent protein kinase was raised in New Zealand white rabbits against a purified (electrophoretically homogeneous) beef heart protein kinase as antigen. Serum globulins were precipitated with ammonium sulfate and antibody activity was characterized employing standard immunodiffusion methods and immunotitration protocols (13). Chromatography. Ion-exchange chromatography was carried out on DEAE-Sepharose columns of 4 x 0.5-cm dimensions. Approximately 1.0 mg of protein was applied per milliliter bed volume and the column was washed with 10 to 20 bed volumes of 20 mM Tris, pH 7.4, 5 mM MgCl,, 5 mM mercaptoethanol, 5% glycerol (v/v). A 0 to 0.35 M NaCl linear gradient was formed in the above buffer and elution continued using a gradient maker. The salt concentrations of the gradient were verified using a Myron LDS Model EP Meter, calibrated with KC1 standards to measure conductivity. Assay of cyclic AMP binding activity. Binding of cyclic [“HIAMP to cell fractions was measured by a modified membrane filter assay as described previously (16). To 20 to 50 pg cell protein were added 25 pmol cyclic [3H]AMP, 16.5 pmol Tris buffer, pH 7.4, 1.7 pmol of theophylline, 2.5 pmol MgCl, in a 0.30-ml volume, and incubated for a minimum of 3 h at 4°C. Five milliliters of cold 0.25 mM Tris buffer, pH 7.4 containing 10 mM MgC& were added to each sample and then filtered using Millipore filters presoaked in this buffer. The samples were washed with an additional 5 ml of buffer, dried, dissolved in scintillation medium, and counted. RESULTS
Substrate Specificity of Nuclear and Cytosol Protein Kinase
Protamine and histone Hl were tested as phosphate acceptors to determine which served as the most efficient exogenous protein substrate for thymocyte protein kinase. As illustrated in Fig. 1, both nuclear and cytosol preparations phosphorylated protamine to a greater extent than histone, regardless of the species origin of the tissue. Also illustrated in Fig. 1 is the protein kinase activity remaining in the nuclear and cytoplasmic preparations after
ONTOGENY
AND MULTIPLICITY
OF LYMPHOID
FIG. 1. Protein kinase levels and substrate specificity of nuclear and cytosol protein kinase in lymphoid cells of rats, calves, and humans. Open bars: protein kinase activity measured with protamine and lo-” M cyclic AMP; hatched bars: protein kinase measured with histone HI as substrate and lo-” M cyclic AMP. The solid bars represent cyclic AMPindependent protein kinase measured with either protamine or histone Hl in the presence of saturating amounts of heat-stable inhibitor and lo-” M cyclic AMP. The listed values are averages of duplicate assays of representative experiments. See text for an evaluation of the reproducibility of the data. S.A. on the ordinate denotes specific activity measured as pmol [32P]phosphate incorporatedimin at 30”Clmg protein.
the addition of saturating concentrations of heat-stable protein kinase inhibitor. The majority (up to 95%) of the phosphorylating activity was inhibited when protamine was used as substrate. The remaining cyclic AMP-independent protein kinase activity is represented by the solid bars in Fig. 1. When histones (either lysine rich or arginine rich or total histones the results are the same, data shown for Hl only) were used as exogenous protein substrates, the relative degree of inhibition was markedly diminished. The total cyclic AMP-independent kinase activities expressed in the presence of inhibitor were comparable when either histone or protamine was used as substrate indicating that in our preparation histones served as more efficient substrates for the cyclic AMP-independent rather than for the cyclic AMP-dependent protein kinase.
CELL PROTEIN
KINASE
187
In order to determine the reproducibility of the basal values shown in Fig. 1, data from several consecutive experiments using thymus cells from 25- to 30-day-old rats were subjected to statistical analysis. The specific activity of the nuclear cyclic AMPdependent protein kinase, measured with protamine, was found to be relatively constant (987 pmol phosphate incorporated/ min/mg protein +202 SD; n = 33), indicating that if loss from the nucleus occurs in preparation it is not appreciably variable. Cytosol protein kinase activity, on the other hand, varied significantly from one preparation to the next (1.3 nmol phosphate incorporated/min/mg protein 20.824 SD; n = 19), indicating either instability of the preparation or inconsistent solubilization of a particulate enzyme, or age-dependent variability even in a span of 5 days. Preliminary data in our laboratory show a significant portion of protein kinase activity of rat thymocytes to be associated with a mitochondrialimicrosomal particulate fraction (15,000 to 105,OOOg pellet). Effects of Cyclic Nucleotides
Both cyclic AMP and cyclic 3’:5’-guanosine monophosphate (cyclic GMP) activated protein kinase in our assay mixtures as shown in a graph of a representative experiment in Fig. 2. Concentrationdependent activation of protein phosphotransferase activity by cyclic AMP in both the nuclear nonhistone fraction and in the cytoplasmic cell fraction was found to be in the range of approximately lop8 to lop5 M cyclic nucleotide. A decrease in enzyme activity was observed when cyclic AMP concentrations exceeded lop5 M. This effect is especially marked in the soluble fraction where a distinct inhibition (below basal values measured in the absence of added cyclic AMP to the assay) was observed (bottom panel in Fig. 2). Cyclic GMP also activated protein kinase in our preparations at concentrations approximately one order of magnitude higher than cyclic AMP. Similar results were found in activation of protein kinase from rabbit skeletal muscle (17) and in lymphocyte preparations (18). Treatment
188
MEDNIEKS.
25 23 21
,* N 1 8
c
NUCLEAR EXTRACT
JUNGMANN.
+-t 0-o
CAMP CGMP
5 t .A’ IO 9
e
7
6
CYTOSOL
5
4
5
+ 4
/T
4kh IO 9
6 -lo,”
[M]
cyclic ’
nucleolide
FIG. 2. Cyclic nucleotide concentration effects on protein kinase activity in the nuclear and cytoplasmic fractions of rat thymus cells. The curve described by crosses (+) represents the effect of cyclic AMP and the curve described by the open circles (0) represents the effects of cyclic GMP at concentrations listed on the abcissa. The ordinate shows 32P incorporation into the acid-insoluble material in aliquots of cell fractions of equal protein concentration.
with a heat-stable inhibitor preparation of the nuclear and cytoplasmic enzyme preparations activated by 1OF and lop5 M
AND
cyclic GMP resulted in variable degrees of reduction of phosphorylative activity (generally in the range of 30 to 60% inhibition). It therefore cannot be determined from our results whether different kinases are activated by cyclic AMP and cyclic GMP or whether activation is due to different binding sites on the same enzyme. The apparent inhibition of protein kinase activity by the low concentrations of cyclic nucleotides in the cytosol may be a function of recurring high initial values (without exogenous cyclic nucleotides-seen in approximately one-third of the experiments). Whether this might be due to selective dissociation-reassociation properties in the absence or at low cyclic nucleotide concentrations is not known. At this time the most reasonable explanation seems that a variability gives false high basal values rather than that low cyclic nucleotide concentrations are causing inhibition of protein kinase activity. Lymphoid Cell Protein Function of Age
KINASE
ACTIVITY
in days 6 10 12 25 50
Number of experiments
IN THYMUS
5 4 6 8 4
Nuclear -CAMP 3.32 5.94 6.64 4.43 3.91
(1.34) (1.21) (0.18) (0.71) (2.30)
as a
I CELLS Specific
Age
Kinase Activity
Studies were carried out using protein kinase preparations from thymocytes of rats and humans of various ages. In rats nuclear protein kinase activity reached maximum levels at about 10 days of age and remained relatively constant thereafter (Table I).
TABLE PROTEIN
SCHWEPPE
OF DEVELOPING activity
RATS
X 102”
fraction*
cytoso1 +cAMP
3.82 9.38 13.42 10.60 9.62
(1.78) (2.10) (2.86) (2.12) (1.91)
-CAMP 9.60 8.27 13.95 18.24 9.74
(4.50) (2.09) (8.24) (3.12) (3.02)
+cAMP 8.74 10.10 21.90 33.57 9.94
(3.10) (2.02) (4.96) (8.75) (3.12)
a Specific activity is measured as the number of pmol [32P]phosphate incorporatedimin at 30”C/mg protein. b Nuclear and cytoplasmic fractions were prepared and assayed for protein kinase activity as described -CAMP and +cAMP indicate assay in the absence under Materials and Methods using protamine as substrate. and in the presence of lOmE M cyclic AMP, respectively. Values are averages of duplicate assays of the listed number of tissue samples. Standard deviations are reported in parentheses after each value.
ONTOGENY
AND MULTIPLICITY
OF LYMPHOID
Cytosol protein kinase reached an activity maximum at approximately 25 days of age. In 50-day-old rats the cytosol protein kinase levels had declined to about half the values observed at 25 days of age. Stimulation by addition of cyclic AMP to the assay is more pronounced and reproducible in nuclear than in cytoplasmic preparations except in preparations from the B-day-old rats when no stimulation was observed. In the cell protein concentration range employed (2 to about 30 ygiassay) mixing experiments indicated no inhibitor activity associated with the young or old rat cell preparations. Results using a heat-stable inhibitor preparation (data no shown) indicate that the fraction of phosphorylative activity due to cyclic AMP-independent protein kinase in both particulate and soluble fractions of the cells either from human or rat thymus of all age groups tested is similar: in the range of lo-30% of the total. Lack of increasing activities by adding cyclic AMP therefore does not mean the absence of cyclic
CELL
PROTEIN
KINASE
189
AMP-dependent protein kinase in these preparations. Cyclic AMP binding studies of the preparations of older rats resulted in patterns similar to those shown in Fig. 3: as in panel B for the nuclear extract and as in panel D in the cytoplasm. In the preparations of the nuclear fraction a significant amount of binding was associated with the column eluate prior to the salt gradient region. These results indicate that although the regulatory subunits are present in these preparations, the enzyme is apparently in the dissociated rather than in the holoenzyme form. This occurs only in preparations of particulate cell fractions and is not a preparational inconsistency since no such binding activity is seen in the wash portion of the rat or beef heart soluble fractions used as type I and type II standards. Dissociation of the cyclic AMP-dependent isozymes into subunits by a protein substrate or by high (0.5 M) NaCl has been demonstrated (19) in other cell systems.
FIG. 3. DEAE-Sepharose elution profiles of protein kinase activity in preparations of lymphoid cells from various species. (A) Nuclear extract from thymocytes of B&day-old rats; (B) rat heart soluble fraction; (C) thymocyte cytosol from 25-day-old rats; (D) beef heart cyclic AMP-dependent protein kinase (Sigma Chemical Co.); (E) nuclear extract from 1%day-old rat thymus; (F) g-year-old human thymus. Open circles, protein kinase activity measured in the absence of cyclic AMP; closed circles, protein kinase activity measured in the presente of IO-” M cyclic AMP; shaded area, cyclic AMP-independent protein kinase activity measured in the presence of saturating amounts of heat-stable inhibitor and lo-” M cyclic AMP. The area denoted by vertical bars topped by triangles in (B) and (D) represents the cyclic [3H]AMP binding profiles. The ordinate denotes counts of 3zP or “H per minute incorporated per fraction assayed.
190
MEDNIEKS,
JUNGMANN,
In humans a decline of cyclic AMPdependent protein kinase activity in nuclear and cytosol cell fractions was apparent in thymocytes of adolescent (16 year old) as compared to juvenile (9 year old) Caucasian males (Table II). The trend of declining activity was maintained in protein kinase preparations of 23-year-old and older males. After the age of 40 the decline in specific activity of cyclic AMP-dependent protein kinase was even more pronounced and the activity decreased to marginally measureable levels. Studies of phytohemagglutinin stimulated blood lymphocytes also show that there is less [32P]phosphate incorporation into endogenous nuclear proteins with increasing age (20) in humans. Chromatographic Characterization Kinase
and Immunochemical of Thymocyte Protein
DEAE-Sepharose chromatography was employed to distinguish protein kinase type I isozyme from the type II isozyme in lymphoid cell cytosol and nuclear extracts. Soluble cyclic AMP-dependent protein kinases from rat heart (type I) and beef heart (type II) were used as standards to TABLE
II
PROTEIN KINASE ACTIVITY IN HUMAN THYMUS LYMPHOID CELLS
Specific activity X 102” Age
(years)
Sex
Nuclear fraction
cytoso1
9 16 23 42 45b 550 69
M M F F M M F
12.62 9.24 2.71 3.00
10.36 7.40 4.20 1.71 1.23 0.06 0.67
0.01
0 As described in Table I. b Insufficient tissue was obtained to carry out the indicated (-) determinations. Nuclear and cytosol fractions were prepared and assayed for protein kinase activity as described under Materials and Methods using protamine as substrate. The values obtained are mean values of duplicate assays of one tissue preparation and assayed in the presence of 1Om6M cyclic AMP.
AND SCHWEPPE
determine the salt gradient elution profiles of these protein kinase isozymes (21). After application of identical amounts of lymphoid cell protein onto the columns, eluted of protein kinase activity was carried out. The elution profiles were compared to the elution profiles of either rat heart (type I) or beef heart (type II) protein kinase. Comparison of the elution profiles (Fig. 3) indicated that nuclear protein from adult rat thymocytes contains mainly the type I isozyme in addition to a small amount of type II protein kinase (Fig. 3A). In contrast, protein kinase activity in the cytosol (Fig. 3C) was predominantly the type II isozyme. Similar results have been obtained using human red blood cells (22) where the type I isozyme was associated with the membrane fraction while the type II isozyme is in the soluble fraction. Cyclic AMP binding data (shaded areas in Figs. 3B and D) indicate that the regulatory subunits are eluted at approximately the same ionic strength as the protein fraction with catalytic activity. Stimulation by cyclic AMP however was relatively small in these regions of the eluate indicating that in these column fractions the enzyme is apparently already in the dissociated form. Since the respective regulatory subunits or the type I and II holoenzymes establish the DEAE election properties the likeliest explanation is that the protein kinase has dissociated within or prior to assay, but not prior to chromatography. The elution profiles of nuclear protein kinase activity from juvenile thymus cells (12-day-old rat, Fig. 3E; g-year-old human, Fig. 3F) show greater chromatographic heterogeneity. Nevertheless, the major peak of activity can be identified as type I isozyme in addition to some type II protein kinase eluting at higher ionic strength. The activity of both protein kinases was readily inhibited by the heat-stable inhibitor and significantly stimulated by the addition of 1O-6 M cyclic AMP to the assay mixture. Rechromatography of the wash peak in Figs. 3E and F results in activity in the type II protein kinase region. Lack of stimulation by cyclic AMP probably reflects that the enzyme is present in the dissociated form.
Immunodiffusion experiments were employed in addition to the studies with the inhibitor protein to distinguish between the cyclic AMP-dependent and cyclic AMPindependent protein kinases and to determine whether similar antigenie sites are present on protein kinases of both type I and type II of various species. Electrophoretically and chromatographically (by Sephadex gel filtration) pure calf and rat thymus cyclic AMP-dependent protein kinase cross-react with purified commercially obtained beef heart enzyme which was used as antigen. Cyclic AMP-independent protein kinase obtained from calf thymus nuclei (21) did not react with the antiserum, corroborating our inhibitor study results.
Thus, positive and negative modulation of intracellular biochemical events by selective activation of site-specific protein kinase isotypes can provide a control mechanism for cell growth. This is demonstrated in lymphoid cells where stimulation with a large dose of a mitogen leads to inhibition of transcriptional processes (32) whereas a low dose promotes cell division and differentiation (31). A decline in protein kinase activity with age in thymus lymphoid cells might well be related to decreases in regulatory control of cell growth. Consequently decreased capacity for immune surveilence can be expressed by increased incidence of proliferative disorders. ACKNOWLEDGMENTS
DISCUSSION
In lymphoid cells changes in cyclic AMP levels have been shown to be associated with differentiative as well as proliferative (23-25) processes. Molecular events responsible for lymphoid cell differentiation as a consequence of stimulation by certain effector agents apparently involve specific T-cell receptors (26) and might well include phosphorylation and functional modification of proteins by cyclic AMP-dependent protein kinase. Some of the biochemical characteristics of cyclic AMP-dependent protein kinase of lymphoid cells are comparable to those of other cell types (3, 13, 27). The relative distribution of protein kinase activity between the nuclear and cytosol cell fractions differs, however, in lymphoid cells when compared to the distribution of protein kinase in other cell types (3, 16, 24, 28) and reflects a specific compartmentalization. The functional significance of cellular site restriction and variation in basal activity as a function of age are not known but our current studies and studies in other laboratories (29, 30) implicate intracellular redistribution of protein kinase as a cyclic AMP-mediated regulatory event. Russell and her associates found that in mitogenstimulated lymphocytes the ensuing biochemical changes can be blocked by activating the type II cyclic AMP-dependent protein kinase at a time when only the type I isozyme is normally activated.
The authors wish to express their appreciation to Ms. Janice Guziec and Ms. Linda Kawano for their expert technical assistance. Thymus tissue from surgery (from individuals free of cancer and not treated with anti-proliferative agents) was obtained in compliance with regulations governing the use of human materials. REFERENCES 1. ROBINSON, G. A., BUTCHER, R. W., AND SUTHERLAND, E. W. (1971) Cyclic AMP, Academic Press, New York. 2. Kuo, J. F., AND GREENGARD. P. (1969) Proc. Nut. Acad. Sci. USA 64, 1349-1353. 3. RUBIN, C. S., AND ROSEN, 0. M. (1975) AWZU. Rev. Biochem. 44, 831-887. 4. LANGAN, T. A. (1973) Advan. Cyclic Nucleotide Res. 3, 99-153. 5. WEDNER, H. J., HOFFER, B. W., BATTENBERG, E., STEINER, A. L., PARKER, C. W., AND BLOOM, F. E. (1972) J. Histochem. Cytochem. 20, 293-295. 6. STEINER, A. L., ONG, S., AND WEDNER, H. J. (1976) Advan. Cyclic Nucleotide Res. 7, 115-153. 7. JOHNSON, E. M., HADDEN, J. W., INOUE, A., AND ALLFREY, V. G. (1975) Biochemistry 14, 3873-3884. 8. KEMP, B. E., FROSCIO, M., ROGERS, A., AND MURRAY, W. W. (1975) Biochem. J. 145, 241-249. 9. MASARACCHIA, R. A., AND WALSH, D. A. (1976) Cancer Res. 36, 3227-3237. 10. KLEIN, M., AND MAKMAN, M. J. (1971) Cell. Physiol. 79, 407-413. 11. PIRAS, M. M., HORENSTEIN, A., AND PIRAS, R. (1977) Enzyme 22, 219-229.
192
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JUNGMANN,
12. HORENSTEIN, A., PIRAS, M. M., MORDOH, J., AND PIRAS, R. (1976) Eq. Cell Res. 101, 260-266. 13. SPIELVOGEL, A. M., MEDNIEKS, M. I., EPPENBERGER, U., AND JUNGMANN, R. A. (1977) Eur. J. Biochem. 73, 199-212. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. WALSH, D. A., ASHBY, C. D., GONZALEZ, C., CALKINS, D., FISHER, E. H., ANDKREBS, E. G. (1971) J. Biol. Chem. 246, 1977-1985. 16. HIESTAND, P. C., EPPENBERGER, U., AND JUNGMANN, R. A. (19’73) Endocrinology 93, 217-230. 17. SODERLING, T. R., HICKENBOTTOM, J. P., REIMANN, E. M., HUNKELER, F. L., WALSH, D. A., AND KREBS, E. G. (1970) J. Biol. Chem. 245, 6317-6328. 18. FARAGO, A., HAZNOS, P., ANTONI, F., AND ROMHANYI, T. (1978) Biochim. Biophys. Acta 538, 493. 19. CORBIN, J. D., KEELY, S. L., AND PARK, C. R. (1975) J. Biol. Chem. 250, 218-225. 20. LUTZ, H., KONERMAN, T., AND ERWINI, M. (1977) Aktuel Gerontol 7, 257-261. 21. CORBIN, J. D., SUGDEN, P. H., LINCOLN, T. M., AND KEELY, S. L. (1977) J. Biol. Chem. 252, 3854-3861. 22. DREYFUSS, G., SCHWARTZ, K. J., AND BLOUT,
AND
23. 24.
25. 26. 27.
28. 29.
30. 31. 32.
33.
SCHWEPPE
E. R. (1978) Proc. Nat. Acad. Sci. USA 75, 5926-5930. KRANIAS, E. G., AND JUNGMANN, R. A. (1978) Biochem. Biophys. Acta 517, 493. BRAUN, W., ISHIZUKA, M., WINCHURCH, R., AND WEBB, D. (1971) Ann. N.Y. Acad. Sci. 185, 417-422. MEDNIEKS, M. I., AND JURAS, D. S. (1975) Immunol. Commun. 4, 99-105. ISHIZUKA, M., GAFNI, M., AND BRAUN, W. (1970) Proc. Sot. Exp. Biol. Med. 134, 963-967. MEDNIEKS, M. I., CRAM, S. L., AND JURAS, D. S. (1978) Biochem. Biophys. Res. Commun. 80, 267-277. LEE, P. C., AND JUNGMANN, R. A. (1975) Biochem. Biophys. Acta 399, 265-276. DE ANGELO, A. B., SCHWEPPE, J. S., JUNGMANN, R. A., HUBER, P., AND EPPENBERGER, U. (1975) Endocrinology 97, 1509-1520. MEDNIEKS, M. I., AND JUNGMANN, R. A. (1978) Advan. Cyclic Nucleotide Res. 12, 760. H. (1977) Eur. J. SCHWOCH, G., AND HILZ, Biochem. 76, 269-276. BYUS, C. V., KLIMPEL, G. R., LUCAS, D. A., AND RUSSEL, D. H. (1978) Mol. Pharmacol. 14, 431-441. HADDEN, J. W., HADDEN, D. M., SAKLIK, J. R., AND COFFEY, R. G. (1976) Proc. Nat. Acad. Sci. USA 73, 1717-1721.
ARCHIVES
Ontogeny MAIJA
and Multiplicity of Cyclic AMP-Dependent in Thymic Lymphoid Cells1
I. MEDNIEKS,
Department
RICHARD
of Biochemistry,
Northwestern
A. JUNGMANN, University
AND
Medical
Protein
JOHN
School,
Chicago,
Kinase
S. SCHWEPPE Illinois
60611
Received January 23, 1979; revised April 12, 1979 Cyclic adenosine 3’:5’-monophosphate-dependent protein kinases were studied in thymus lymphoid cells and were found to be similar to their counterparts in other tissues with respect to substrate preference and concentration dependence. A previously not identified, restrictive subcellular compartmentalization of the protein kinase isozymes was found: Type I was predominantly present in the nucleus of adult and juvenile human and rat thymus cells, whereas the type II kinase was restricted to the cytosol fraction of unstimulated cells. Additionally, a decline in the specific activity of protein kinase was progressive with increasing age of the animal and distinct from the general observation that lymphoid cell numbers decrease with age. These findings may be correlated with age-dependent immunodeficiencies and perhaps have functional significance in the regulatory role of protein phosphorylation in lymphoid cell activation.
Lymphoid cells, as do virtually all mammalian cells, contain cyclic adenosine 3’:5’-monophosphate (cyclic AMP) and respond to regulation by a number of environmental stimuli with changes in cyclic nucleotide levels (1). Physiological events which are mediated by cyclic nucleotides in eucaryotic cells involve phosphorylation of protein substrates (2-4) by cyclic AMP-dependent protein kinase (EC 2.7.1.37). Using immunocytochemical techniques (5, 6) distinct intracellular cyclic AMP pools have been identified in lymphocytes, and nuclear protein phosphorylation has been shown to be related to changes in cyclic AMP pools in these cells (7, 8). Information regarding the biochemical characteristics of cyclic AMP-dependent protein kinase in lymphoid cells is therefore relevant to understanding nuclear events which participate in the regulation of immune responses. The present study examines biochemical characteristics of both nuclear and cytoplasmic cyclic AMP-dependent protein kinases and some aspects of its ontogeny 1 This study was in part supported by the Research and Education Fund, Northwestern Memorial Hospital.
in a selected population of lymphoid cells. Thymic tissue has been used in similar studies (9, 10) and provides a convenient source of cells which are relatively free of contaminants in the form of other cell types such as red and white blood cells or fat and connective tissue components. Cell homogeneity becomes a critical consideration when the presence and distribution of protein kinase isozymes is measured. Also, since some biochemical data are available from normal and transformed T-cells (11, 12), comparisons are feasible regarding the intracellular distribution and activity of soluble and particulate protein kinases. It is apparent from our studies that cyclic AMP-dependent protein kinases exhibit a biochemical constancy in thymic cells from humans and from various animals. This finding provides a suitable basis for studying molecular mechanisms of lymphoid cell stimulation in an animal model and might be applied to examining analogous normal and neoplastic human lymphoid cell systems. MATERIALS Tissue
Fresh 185
preparation
thymus
AND METHODS and
tissue
subcellular
fractionation.
was placed
in phosphate
0003-9861/79/110185-O&$02.00/0
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction
in any form reserved.
186
MEDNIEKS.
JUNGMANN.
buffered saline (PBS),’ pH 7.5 (GIBCO, Grand Island, N. Y.) and kept on ice until use. Connective tissue, visible blood vessels, fat, and parathymic lymph nodes were stripped away and thymus cells were separated by gently pressing the tissue through a number 80 mesh (60 pm) stainless-steel screen (Fisher Scientific Co.) with a rubber tipped plunger of a ~-CC syringe. The cells were pelleted using a desk-top clinical centrifuge, resuspended in PBS, strained through gauze, and collected by centrifugation. The washed cells were homogenized in 0.25 M sucrose, 2 mM CaCl,, 4 mM theophylline, 50 mM Tris, pH 7.4 buffer. A crude nuclear pellet was obtained by centrifugation for 20 min at 9OOg. Purified nuclei were obtained by subsequent centrifugation at 105,OOOg for 1 h through 2.0 M sucrose, containing 2 mM CaCI,, 50 mM Tris, pH 7.4. The purified nuclear pellet was rinsed with PBS and extracted with a 0.35 M NaCl, 50 mM MgCI,, 3 mM dithiothreitol, 20 mM Tris, pH 7.4 solution. The nuclear extract and the cytosol (obtained by centrifuging the 900s supernatant at 105,OOOg for 1 h) were then dialyzed against two changes of 5 mM MgCl,, 5 mM mercaptoethanol, 5% glycerol, in 20 mM Tris, pH 7.4. The dialyzed solutions were cleared of precipitated material by centrifugation and subsequently used for enzyme assays and DEAE-Sepharose (Pharmacia) chromatography. Determination of protein kinase activity. The assay was carried out as described previously (13) in a volume of 0.25 ml containing 50 p,g of protamine sulfate or histone Hl, 5 mM a-glycerol phosphate buffer, pH 7.5, 10 mM [y-3ZP]ATP (0.5 &i) without and with cyclic AMP (10m6M in assaying cell fractions and lo-’ M in assaying column fractions), as indicated. Incubation was carried out for 10 min at 30°C and the reaction was stopped by adding 2 ml of 15% trichloroacetic acid (TCA) containing 1% sodium dodecyl sulfate (SDS). The samples were filtered using Millipore filters and washed with the TCA-SDS solution. Radioactivity was measured in a Packard Tri-Carb liquid scintillation counter (Model 3375). Assay reagents were purchased from Sigma Chemical Company. Protein content was determined by the Lowry method (14). Protein kinase activity was measured in cell fractions containing between 20 and 50 fig protein per assay, a range in which phosphate incorporation was linear for all tissues tested. Under these experimental conditions incorporation of [“‘PIphosphate into substrate protein was linear over a 20-min incubation period. Preparation of inhibitor and of antiserum to protein kinase. A heat-stable protein kinase inhibitor preparation was obtained according to the method of * Abbreviations used: PBS, Phosphate buffered saline; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate.
AND SCHWEPPE Walsh et al. (15) from rabbit skeletal muscle. The inhibitor was purified on DEAE-cellulose and used in 5 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA at a concentration of 1 mg of proteinlmi. This preparation was used in conjunction with immunochemical methods to establish the extent of cyclic AMP-independent phosphorylation in our preparations. Antiserum to cyclic AMP-dependent protein kinase was raised in New Zealand white rabbits against a purified (electrophoretically homogeneous) beef heart protein kinase as antigen. Serum globulins were precipitated with ammonium sulfate and antibody activity was characterized employing standard immunodiffusion methods and immunotitration protocols (13). Chromatography. Ion-exchange chromatography was carried out on DEAE-Sepharose columns of 4 x 0.5-cm dimensions. Approximately 1.0 mg of protein was applied per milliliter bed volume and the column was washed with 10 to 20 bed volumes of 20 mM Tris, pH 7.4, 5 mM MgCl,, 5 mM mercaptoethanol, 5% glycerol (v/v). A 0 to 0.35 M NaCl linear gradient was formed in the above buffer and elution continued using a gradient maker. The salt concentrations of the gradient were verified using a Myron LDS Model EP Meter, calibrated with KC1 standards to measure conductivity. Assay of cyclic AMP binding activity. Binding of cyclic [“HIAMP to cell fractions was measured by a modified membrane filter assay as described previously (16). To 20 to 50 pg cell protein were added 25 pmol cyclic [3H]AMP, 16.5 pmol Tris buffer, pH 7.4, 1.7 pmol of theophylline, 2.5 pmol MgCl, in a 0.30-ml volume, and incubated for a minimum of 3 h at 4°C. Five milliliters of cold 0.25 mM Tris buffer, pH 7.4 containing 10 mM MgC& were added to each sample and then filtered using Millipore filters presoaked in this buffer. The samples were washed with an additional 5 ml of buffer, dried, dissolved in scintillation medium, and counted. RESULTS
Substrate Specificity of Nuclear and Cytosol Protein Kinase
Protamine and histone Hl were tested as phosphate acceptors to determine which served as the most efficient exogenous protein substrate for thymocyte protein kinase. As illustrated in Fig. 1, both nuclear and cytosol preparations phosphorylated protamine to a greater extent than histone, regardless of the species origin of the tissue. Also illustrated in Fig. 1 is the protein kinase activity remaining in the nuclear and cytoplasmic preparations after
ONTOGENY
AND MULTIPLICITY
OF LYMPHOID
FIG. 1. Protein kinase levels and substrate specificity of nuclear and cytosol protein kinase in lymphoid cells of rats, calves, and humans. Open bars: protein kinase activity measured with protamine and lo-” M cyclic AMP; hatched bars: protein kinase measured with histone HI as substrate and lo-” M cyclic AMP. The solid bars represent cyclic AMPindependent protein kinase measured with either protamine or histone Hl in the presence of saturating amounts of heat-stable inhibitor and lo-” M cyclic AMP. The listed values are averages of duplicate assays of representative experiments. See text for an evaluation of the reproducibility of the data. S.A. on the ordinate denotes specific activity measured as pmol [32P]phosphate incorporatedimin at 30”Clmg protein.
the addition of saturating concentrations of heat-stable protein kinase inhibitor. The majority (up to 95%) of the phosphorylating activity was inhibited when protamine was used as substrate. The remaining cyclic AMP-independent protein kinase activity is represented by the solid bars in Fig. 1. When histones (either lysine rich or arginine rich or total histones the results are the same, data shown for Hl only) were used as exogenous protein substrates, the relative degree of inhibition was markedly diminished. The total cyclic AMP-independent kinase activities expressed in the presence of inhibitor were comparable when either histone or protamine was used as substrate indicating that in our preparation histones served as more efficient substrates for the cyclic AMP-independent rather than for the cyclic AMP-dependent protein kinase.
CELL PROTEIN
KINASE
187
In order to determine the reproducibility of the basal values shown in Fig. 1, data from several consecutive experiments using thymus cells from 25- to 30-day-old rats were subjected to statistical analysis. The specific activity of the nuclear cyclic AMPdependent protein kinase, measured with protamine, was found to be relatively constant (987 pmol phosphate incorporated/ min/mg protein +202 SD; n = 33), indicating that if loss from the nucleus occurs in preparation it is not appreciably variable. Cytosol protein kinase activity, on the other hand, varied significantly from one preparation to the next (1.3 nmol phosphate incorporated/min/mg protein 20.824 SD; n = 19), indicating either instability of the preparation or inconsistent solubilization of a particulate enzyme, or age-dependent variability even in a span of 5 days. Preliminary data in our laboratory show a significant portion of protein kinase activity of rat thymocytes to be associated with a mitochondrialimicrosomal particulate fraction (15,000 to 105,OOOg pellet). Effects of Cyclic Nucleotides
Both cyclic AMP and cyclic 3’:5’-guanosine monophosphate (cyclic GMP) activated protein kinase in our assay mixtures as shown in a graph of a representative experiment in Fig. 2. Concentrationdependent activation of protein phosphotransferase activity by cyclic AMP in both the nuclear nonhistone fraction and in the cytoplasmic cell fraction was found to be in the range of approximately lop8 to lop5 M cyclic nucleotide. A decrease in enzyme activity was observed when cyclic AMP concentrations exceeded lop5 M. This effect is especially marked in the soluble fraction where a distinct inhibition (below basal values measured in the absence of added cyclic AMP to the assay) was observed (bottom panel in Fig. 2). Cyclic GMP also activated protein kinase in our preparations at concentrations approximately one order of magnitude higher than cyclic AMP. Similar results were found in activation of protein kinase from rabbit skeletal muscle (17) and in lymphocyte preparations (18). Treatment
188
MEDNIEKS.
25 23 21
,* N 1 8
c
NUCLEAR EXTRACT
JUNGMANN.
+-t 0-o
CAMP CGMP
5 t .A’ IO 9
e
7
6
CYTOSOL
5
4
5
+ 4
/T
4kh IO 9
6 -lo,”
[M]
cyclic ’
nucleolide
FIG. 2. Cyclic nucleotide concentration effects on protein kinase activity in the nuclear and cytoplasmic fractions of rat thymus cells. The curve described by crosses (+) represents the effect of cyclic AMP and the curve described by the open circles (0) represents the effects of cyclic GMP at concentrations listed on the abcissa. The ordinate shows 32P incorporation into the acid-insoluble material in aliquots of cell fractions of equal protein concentration.
with a heat-stable inhibitor preparation of the nuclear and cytoplasmic enzyme preparations activated by 1OF and lop5 M
AND
cyclic GMP resulted in variable degrees of reduction of phosphorylative activity (generally in the range of 30 to 60% inhibition). It therefore cannot be determined from our results whether different kinases are activated by cyclic AMP and cyclic GMP or whether activation is due to different binding sites on the same enzyme. The apparent inhibition of protein kinase activity by the low concentrations of cyclic nucleotides in the cytosol may be a function of recurring high initial values (without exogenous cyclic nucleotides-seen in approximately one-third of the experiments). Whether this might be due to selective dissociation-reassociation properties in the absence or at low cyclic nucleotide concentrations is not known. At this time the most reasonable explanation seems that a variability gives false high basal values rather than that low cyclic nucleotide concentrations are causing inhibition of protein kinase activity. Lymphoid Cell Protein Function of Age
KINASE
ACTIVITY
in days 6 10 12 25 50
Number of experiments
IN THYMUS
5 4 6 8 4
Nuclear -CAMP 3.32 5.94 6.64 4.43 3.91
(1.34) (1.21) (0.18) (0.71) (2.30)
as a
I CELLS Specific
Age
Kinase Activity
Studies were carried out using protein kinase preparations from thymocytes of rats and humans of various ages. In rats nuclear protein kinase activity reached maximum levels at about 10 days of age and remained relatively constant thereafter (Table I).
TABLE PROTEIN
SCHWEPPE
OF DEVELOPING activity
RATS
X 102”
fraction*
cytoso1 +cAMP
3.82 9.38 13.42 10.60 9.62
(1.78) (2.10) (2.86) (2.12) (1.91)
-CAMP 9.60 8.27 13.95 18.24 9.74
(4.50) (2.09) (8.24) (3.12) (3.02)
+cAMP 8.74 10.10 21.90 33.57 9.94
(3.10) (2.02) (4.96) (8.75) (3.12)
a Specific activity is measured as the number of pmol [32P]phosphate incorporatedimin at 30”C/mg protein. b Nuclear and cytoplasmic fractions were prepared and assayed for protein kinase activity as described -CAMP and +cAMP indicate assay in the absence under Materials and Methods using protamine as substrate. and in the presence of lOmE M cyclic AMP, respectively. Values are averages of duplicate assays of the listed number of tissue samples. Standard deviations are reported in parentheses after each value.
ONTOGENY
AND MULTIPLICITY
OF LYMPHOID
Cytosol protein kinase reached an activity maximum at approximately 25 days of age. In 50-day-old rats the cytosol protein kinase levels had declined to about half the values observed at 25 days of age. Stimulation by addition of cyclic AMP to the assay is more pronounced and reproducible in nuclear than in cytoplasmic preparations except in preparations from the B-day-old rats when no stimulation was observed. In the cell protein concentration range employed (2 to about 30 ygiassay) mixing experiments indicated no inhibitor activity associated with the young or old rat cell preparations. Results using a heat-stable inhibitor preparation (data no shown) indicate that the fraction of phosphorylative activity due to cyclic AMP-independent protein kinase in both particulate and soluble fractions of the cells either from human or rat thymus of all age groups tested is similar: in the range of lo-30% of the total. Lack of increasing activities by adding cyclic AMP therefore does not mean the absence of cyclic
CELL
PROTEIN
KINASE
189
AMP-dependent protein kinase in these preparations. Cyclic AMP binding studies of the preparations of older rats resulted in patterns similar to those shown in Fig. 3: as in panel B for the nuclear extract and as in panel D in the cytoplasm. In the preparations of the nuclear fraction a significant amount of binding was associated with the column eluate prior to the salt gradient region. These results indicate that although the regulatory subunits are present in these preparations, the enzyme is apparently in the dissociated rather than in the holoenzyme form. This occurs only in preparations of particulate cell fractions and is not a preparational inconsistency since no such binding activity is seen in the wash portion of the rat or beef heart soluble fractions used as type I and type II standards. Dissociation of the cyclic AMP-dependent isozymes into subunits by a protein substrate or by high (0.5 M) NaCl has been demonstrated (19) in other cell systems.
FIG. 3. DEAE-Sepharose elution profiles of protein kinase activity in preparations of lymphoid cells from various species. (A) Nuclear extract from thymocytes of B&day-old rats; (B) rat heart soluble fraction; (C) thymocyte cytosol from 25-day-old rats; (D) beef heart cyclic AMP-dependent protein kinase (Sigma Chemical Co.); (E) nuclear extract from 1%day-old rat thymus; (F) g-year-old human thymus. Open circles, protein kinase activity measured in the absence of cyclic AMP; closed circles, protein kinase activity measured in the presente of IO-” M cyclic AMP; shaded area, cyclic AMP-independent protein kinase activity measured in the presence of saturating amounts of heat-stable inhibitor and lo-” M cyclic AMP. The area denoted by vertical bars topped by triangles in (B) and (D) represents the cyclic [3H]AMP binding profiles. The ordinate denotes counts of 3zP or “H per minute incorporated per fraction assayed.
190
MEDNIEKS,
JUNGMANN,
In humans a decline of cyclic AMPdependent protein kinase activity in nuclear and cytosol cell fractions was apparent in thymocytes of adolescent (16 year old) as compared to juvenile (9 year old) Caucasian males (Table II). The trend of declining activity was maintained in protein kinase preparations of 23-year-old and older males. After the age of 40 the decline in specific activity of cyclic AMP-dependent protein kinase was even more pronounced and the activity decreased to marginally measureable levels. Studies of phytohemagglutinin stimulated blood lymphocytes also show that there is less [32P]phosphate incorporation into endogenous nuclear proteins with increasing age (20) in humans. Chromatographic Characterization Kinase
and Immunochemical of Thymocyte Protein
DEAE-Sepharose chromatography was employed to distinguish protein kinase type I isozyme from the type II isozyme in lymphoid cell cytosol and nuclear extracts. Soluble cyclic AMP-dependent protein kinases from rat heart (type I) and beef heart (type II) were used as standards to TABLE
II
PROTEIN KINASE ACTIVITY IN HUMAN THYMUS LYMPHOID CELLS
Specific activity X 102” Age
(years)
Sex
Nuclear fraction
cytoso1
9 16 23 42 45b 550 69
M M F F M M F
12.62 9.24 2.71 3.00
10.36 7.40 4.20 1.71 1.23 0.06 0.67
0.01
0 As described in Table I. b Insufficient tissue was obtained to carry out the indicated (-) determinations. Nuclear and cytosol fractions were prepared and assayed for protein kinase activity as described under Materials and Methods using protamine as substrate. The values obtained are mean values of duplicate assays of one tissue preparation and assayed in the presence of 1Om6M cyclic AMP.
AND SCHWEPPE
determine the salt gradient elution profiles of these protein kinase isozymes (21). After application of identical amounts of lymphoid cell protein onto the columns, eluted of protein kinase activity was carried out. The elution profiles were compared to the elution profiles of either rat heart (type I) or beef heart (type II) protein kinase. Comparison of the elution profiles (Fig. 3) indicated that nuclear protein from adult rat thymocytes contains mainly the type I isozyme in addition to a small amount of type II protein kinase (Fig. 3A). In contrast, protein kinase activity in the cytosol (Fig. 3C) was predominantly the type II isozyme. Similar results have been obtained using human red blood cells (22) where the type I isozyme was associated with the membrane fraction while the type II isozyme is in the soluble fraction. Cyclic AMP binding data (shaded areas in Figs. 3B and D) indicate that the regulatory subunits are eluted at approximately the same ionic strength as the protein fraction with catalytic activity. Stimulation by cyclic AMP however was relatively small in these regions of the eluate indicating that in these column fractions the enzyme is apparently already in the dissociated form. Since the respective regulatory subunits or the type I and II holoenzymes establish the DEAE election properties the likeliest explanation is that the protein kinase has dissociated within or prior to assay, but not prior to chromatography. The elution profiles of nuclear protein kinase activity from juvenile thymus cells (12-day-old rat, Fig. 3E; g-year-old human, Fig. 3F) show greater chromatographic heterogeneity. Nevertheless, the major peak of activity can be identified as type I isozyme in addition to some type II protein kinase eluting at higher ionic strength. The activity of both protein kinases was readily inhibited by the heat-stable inhibitor and significantly stimulated by the addition of 1O-6 M cyclic AMP to the assay mixture. Rechromatography of the wash peak in Figs. 3E and F results in activity in the type II protein kinase region. Lack of stimulation by cyclic AMP probably reflects that the enzyme is present in the dissociated form.
Immunodiffusion experiments were employed in addition to the studies with the inhibitor protein to distinguish between the cyclic AMP-dependent and cyclic AMPindependent protein kinases and to determine whether similar antigenie sites are present on protein kinases of both type I and type II of various species. Electrophoretically and chromatographically (by Sephadex gel filtration) pure calf and rat thymus cyclic AMP-dependent protein kinase cross-react with purified commercially obtained beef heart enzyme which was used as antigen. Cyclic AMP-independent protein kinase obtained from calf thymus nuclei (21) did not react with the antiserum, corroborating our inhibitor study results.
Thus, positive and negative modulation of intracellular biochemical events by selective activation of site-specific protein kinase isotypes can provide a control mechanism for cell growth. This is demonstrated in lymphoid cells where stimulation with a large dose of a mitogen leads to inhibition of transcriptional processes (32) whereas a low dose promotes cell division and differentiation (31). A decline in protein kinase activity with age in thymus lymphoid cells might well be related to decreases in regulatory control of cell growth. Consequently decreased capacity for immune surveilence can be expressed by increased incidence of proliferative disorders. ACKNOWLEDGMENTS
DISCUSSION
In lymphoid cells changes in cyclic AMP levels have been shown to be associated with differentiative as well as proliferative (23-25) processes. Molecular events responsible for lymphoid cell differentiation as a consequence of stimulation by certain effector agents apparently involve specific T-cell receptors (26) and might well include phosphorylation and functional modification of proteins by cyclic AMP-dependent protein kinase. Some of the biochemical characteristics of cyclic AMP-dependent protein kinase of lymphoid cells are comparable to those of other cell types (3, 13, 27). The relative distribution of protein kinase activity between the nuclear and cytosol cell fractions differs, however, in lymphoid cells when compared to the distribution of protein kinase in other cell types (3, 16, 24, 28) and reflects a specific compartmentalization. The functional significance of cellular site restriction and variation in basal activity as a function of age are not known but our current studies and studies in other laboratories (29, 30) implicate intracellular redistribution of protein kinase as a cyclic AMP-mediated regulatory event. Russell and her associates found that in mitogenstimulated lymphocytes the ensuing biochemical changes can be blocked by activating the type II cyclic AMP-dependent protein kinase at a time when only the type I isozyme is normally activated.
The authors wish to express their appreciation to Ms. Janice Guziec and Ms. Linda Kawano for their expert technical assistance. Thymus tissue from surgery (from individuals free of cancer and not treated with anti-proliferative agents) was obtained in compliance with regulations governing the use of human materials. REFERENCES 1. ROBINSON, G. A., BUTCHER, R. W., AND SUTHERLAND, E. W. (1971) Cyclic AMP, Academic Press, New York. 2. Kuo, J. F., AND GREENGARD. P. (1969) Proc. Nut. Acad. Sci. USA 64, 1349-1353. 3. RUBIN, C. S., AND ROSEN, 0. M. (1975) AWZU. Rev. Biochem. 44, 831-887. 4. LANGAN, T. A. (1973) Advan. Cyclic Nucleotide Res. 3, 99-153. 5. WEDNER, H. J., HOFFER, B. W., BATTENBERG, E., STEINER, A. L., PARKER, C. W., AND BLOOM, F. E. (1972) J. Histochem. Cytochem. 20, 293-295. 6. STEINER, A. L., ONG, S., AND WEDNER, H. J. (1976) Advan. Cyclic Nucleotide Res. 7, 115-153. 7. JOHNSON, E. M., HADDEN, J. W., INOUE, A., AND ALLFREY, V. G. (1975) Biochemistry 14, 3873-3884. 8. KEMP, B. E., FROSCIO, M., ROGERS, A., AND MURRAY, W. W. (1975) Biochem. J. 145, 241-249. 9. MASARACCHIA, R. A., AND WALSH, D. A. (1976) Cancer Res. 36, 3227-3237. 10. KLEIN, M., AND MAKMAN, M. J. (1971) Cell. Physiol. 79, 407-413. 11. PIRAS, M. M., HORENSTEIN, A., AND PIRAS, R. (1977) Enzyme 22, 219-229.
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12. HORENSTEIN, A., PIRAS, M. M., MORDOH, J., AND PIRAS, R. (1976) Eq. Cell Res. 101, 260-266. 13. SPIELVOGEL, A. M., MEDNIEKS, M. I., EPPENBERGER, U., AND JUNGMANN, R. A. (1977) Eur. J. Biochem. 73, 199-212. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. WALSH, D. A., ASHBY, C. D., GONZALEZ, C., CALKINS, D., FISHER, E. H., ANDKREBS, E. G. (1971) J. Biol. Chem. 246, 1977-1985. 16. HIESTAND, P. C., EPPENBERGER, U., AND JUNGMANN, R. A. (19’73) Endocrinology 93, 217-230. 17. SODERLING, T. R., HICKENBOTTOM, J. P., REIMANN, E. M., HUNKELER, F. L., WALSH, D. A., AND KREBS, E. G. (1970) J. Biol. Chem. 245, 6317-6328. 18. FARAGO, A., HAZNOS, P., ANTONI, F., AND ROMHANYI, T. (1978) Biochim. Biophys. Acta 538, 493. 19. CORBIN, J. D., KEELY, S. L., AND PARK, C. R. (1975) J. Biol. Chem. 250, 218-225. 20. LUTZ, H., KONERMAN, T., AND ERWINI, M. (1977) Aktuel Gerontol 7, 257-261. 21. CORBIN, J. D., SUGDEN, P. H., LINCOLN, T. M., AND KEELY, S. L. (1977) J. Biol. Chem. 252, 3854-3861. 22. DREYFUSS, G., SCHWARTZ, K. J., AND BLOUT,
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E. R. (1978) Proc. Nat. Acad. Sci. USA 75, 5926-5930. KRANIAS, E. G., AND JUNGMANN, R. A. (1978) Biochem. Biophys. Acta 517, 493. BRAUN, W., ISHIZUKA, M., WINCHURCH, R., AND WEBB, D. (1971) Ann. N.Y. Acad. Sci. 185, 417-422. MEDNIEKS, M. I., AND JURAS, D. S. (1975) Immunol. Commun. 4, 99-105. ISHIZUKA, M., GAFNI, M., AND BRAUN, W. (1970) Proc. Sot. Exp. Biol. Med. 134, 963-967. MEDNIEKS, M. I., CRAM, S. L., AND JURAS, D. S. (1978) Biochem. Biophys. Res. Commun. 80, 267-277. LEE, P. C., AND JUNGMANN, R. A. (1975) Biochem. Biophys. Acta 399, 265-276. DE ANGELO, A. B., SCHWEPPE, J. S., JUNGMANN, R. A., HUBER, P., AND EPPENBERGER, U. (1975) Endocrinology 97, 1509-1520. MEDNIEKS, M. I., AND JUNGMANN, R. A. (1978) Advan. Cyclic Nucleotide Res. 12, 760. H. (1977) Eur. J. SCHWOCH, G., AND HILZ, Biochem. 76, 269-276. BYUS, C. V., KLIMPEL, G. R., LUCAS, D. A., AND RUSSEL, D. H. (1978) Mol. Pharmacol. 14, 431-441. HADDEN, J. W., HADDEN, D. M., SAKLIK, J. R., AND COFFEY, R. G. (1976) Proc. Nat. Acad. Sci. USA 73, 1717-1721.
