Printed in U.S.A.. Cyclic AMP-dependent Protein Kinase Type I Mediates the Inhibitory. Effects of 3',5'-Cyclic Adenosine Monophosphate on Cell Replication.
Vol. 267,No. 22, Issue of August 5, pp. 15707-15714,1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY (c) 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclic AMP-dependent Protein Kinase TypeI Mediates the Inhibitory Effects of 3’,5’-Cyclic Adenosine Monophosphate on CellReplication in Human T Lymphocytes* (Received for publication, January 23, 1992)
Bjmn S. SkHlhegg$$, Brynjar F. Landmarkll, Stein 0. D~skelandll, Vidar Hanssonll, Tor Lea$, and Tore Jahnsenll From the $Znstitute of Immunology and Rheumatology, Rikshospitalet, 0027 Oslo 1, Norway, the 7Znstituteof Medical Biochemistrv. Uniuersitv of Oslo. P. 0. Box 11 12 Blindern, 0317 Oslo, Norway, and the (1 Department of Anatomy, Bergen, N-5009 Bergen, Norway Uniuersity if
Human T lymphocytes were used as a model system to study the expression and roles of CAMP-dependent protein kinase isozymes (cAKI and cAKII) in CAMPinduced inhibitionof cell replication. Human peripheral blood T lymphocytes expressed mRNA for the a-subforms (RI, and RII,) of the regulatory subunitsof cAKI and cAKII and for thea- and &subforms (C, and C,) of the catalytic subunits of cAK. At the protein level, RI, represented approximately 7 5 % of the totalR subunit activity, whereas RII, (phospho anddephospho forms) accountedfor the remaining 25%. RII, was not detected at either the mRNA or the protein level. The RI, protein was mainly (>75%) cytosolic, whereas RII, was almost exclusively (>go%) particulate associated. Treatment of proliferating T lymphocytes (activated through theCD3 cellsurface marker) with 10 different cAMP analogs demonstratedthat all inhibited cell replication ina concentration-dependent manner. Thepotency (as measured by the concentration giving 50% inhibition, ICbo) of the cAMP analogs ranged from 30 PM for 8-chlorophenylthio-CAMP to 1100 PM for 8piperidino-CAMP. A cAMP analog pair directed toactivate cAKI (8-aminohexylamino-CAMP and 8-piperidino-CAMP) synergized in the inhibition of T lymphocyte proliferation, whereas a cAKI1-directed cAMP analogpair (8-chlorophenylthio-CAMP and iV-benzoyl-CAMP) did not. We conclude that activation of cAKI is sufficient to inhibit T lymphocyte proliferation. The membrane-bound cAKII may mediate cAMP actions not related tocell replication.
role in the regulation of DNA replication in anumber of cells, including T lymplnocytes (1). Two major types of mammalian cAK (cAKI and cAKII) are described, both of which are isozymes consisting of two catalytic (C) subunits and a regulatory (R) subunit dimer (2). cAKI and cAKI1 show different affinities for DEAE-cellulose, due to differences in the R subunits, designated RI and RII. RI and RII differ in molecular weight, antigenicity, amino acid sequence, their ability to be autophosphorylated, and their affinity for cAMP analogs (3). More recent investigations have documented isoforms of the RI and RII subunits, as well as for the C subunits. The R subunits are designated RI, (4-6), RIB (7), RII, (8), and RII, (9), whereas the C subunits are designated C, (10, ll),CB(12), and C, (13). The RI,, RII,, and C, subunits are expressed in most tissues (3), whereas the RIB and C, are mainly found in the brain and testis (7, 14). The RII, is most abundant in the brain, ovary, and testis (15-17), whereas the C, has so far only been found in the testis (13). Activation ofcAK occurs upon binding of four cAMP molecules, two per R monomer, where the binding takes place on two asymmetric sites (designated A and B) (18, 19) in a positive cooperative fashion (20). This dissociates the holoenzyme, releasing active Csubunit and dimers of the R subunits (21). It has been shown that cAMP analogs with selectivity for site A and B, respectively, can complement each other in the activation of cAK (22, 23) and that analog pairs by synergistic action also can selectively activate cAKI and cAKII in the intactcell (24, 25). DNA replication in resting T lymphocytes can be initiated through a diverse group of cell surface receptors, among them the T-au antigen-CD3 receptor complex (TCR-CD3) (26). Intracellular elevation of cAMP with a subsequent activa- Several reports have demonstratedcAMP to inhibit this tion of CAMP-dependent protein kinase (cAK)‘ plays a key process (27-30). To what extent cAKI or cAKII mediates these effects in T lymphocytes (or in other cells) is still * This work was supported by the Norwegian Cancer Society, the unclear. Experiments on peripheral blood lymphocytes sugNorwegian Research Council for Science and the Humanities, Torst- gested that the mitogen concanavalin A selectively activated edsGrant,the Anders JahresFoundation for the Promotion of cAKI, whereas W,@-dibutyryl-CAMP activated cAKII as well Science, and the Nordic Insulin Foundation Committee. The costs of (31). This raised the question of whether the two isozymes publication of this article were defrayed in part by the payment of might mediate different effects on cell replication in these page charges. This article must therefore be hereby marked “aduertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate cells. However, more recent reports have concluded that both isozymes convey inhibitory signals in lymphocytes (1). this fact. f To whom correspondence should be addressed Institute of ImSince a proper characterizationof lymphocyte cAK is lackmunology and Rheumatology, Rikshospitalet, 0027 Oslo, Norway. ing and because of the conflicting results regarding expression, The abbreviations used are: cAK, cyclic AMP-dependent protein localization, and function of cAKI and cAKII, we have underkinase; cAKI and cAKII, cAK type I and 11; RI,,, RIB,RII-, and RIIB, isoforms of the regulatory subunits of cAK; C,, Cp, and C,, isoforms taken acharacterization of the kinase in highly purified of the catalytic subunit of cAK; kb, kilobase(s); SDS, sodium dodecyl peripheral blood T lymphocytes. In addition, we have used sulfate; PAGE, polyacrylamide gel electrophoresis. site-selective cAMP analogs to synergistically activate cAKI
15707
15708
Cyclic A ~ P - ~ e p e nProtein ~ e n ~Kinase in Human T ~ymphocyte~
and cAKII in intact T lymphocytes to specifically elucidate the potential of each isozyme as modulators of cell replication, MATERIALS AND METHODS
Purif~ationof Peripheral Blood T ~ymphocytes-Human blood lymphocytes were isolated from platelet-depleted buffy coats of healthy normal blood donors (Norwegian National Hospital, Blood Bank). Mononuclear cells were isolated according to the method of Beyum et al. (32). Further purification of lymphocytes was accomplished by incubating the cells in plastic culture flasks (5 X lo6 cells/ ml of RPMI 1640 culture medium containing 5% fetal calf serum). Nonadherent cells were decanted into new plastic culture flasks and incubated over night a t a concentrationof 1X IO6cells/ml of medium. Thereafter, the remaining nonadherent cells were incubated for 30 min at 4 "C with antibodies against the CD37, CD19, CD56 and the major histocompatibility complex class I1 markers (antibodies from clones HH1 (33), AB-1 (34), NKH-1 (Coulter Immunology, Hialeah, FL), and HKB-1 (anti-DP, DQ, and DR, respectively (35)). Cells recognized by the antibodies were removed after incubation (30 min at 4 "C) with magnetic beads coated with sheep anti-mouse IgG (DYNAL, Dynabeads "450, Cat. no. 11002) and exposure to a magnet (36). The purity of the T lymphocytes was routinely investigated by flow cytometry (fluorescence-activated cell sorting analyzer, Becton Dickinson, Sunnyvale, CA) using fluorescein isothiocyanateconjugated antibodies (Becton Dickinson and DAKO) against the above-mentioned markers, as well as themarkers for CD14 and CD25. Using this purification method, we obtained cell populations of more than 98% T lymphocytes. Northern Blot Analysis-Total RNA was isolated from T lymphocytes (6 X lo7 cells) using the guanidinium isothiocyanate method of Chirgwin et al. (37). Isolated RNA wasanalyzed by Northern blotting, as previously described (38). Complementary DNA Probes for Northern Blots-Probing Northern filters with cDNAs for the human R subunits RI, (l6), RIB (14), RH,, (39), and RIIB (40) and the human C subunits C,, Cp, and C, (13) was accomplished as previously described (38). Preparation of Soluble Cell Extracts-Cell extracts used for measuring phosphotransferase activity, cAMP binding, photoaffinity labeling, and immunolabeling (see below) were prepared by homogenizing 5 X lo7 T lymphocytes 3 X 15 s (Ultra Turrax, full speed) in 1 ml of buffer containing 10 mM phosphate, pH 6.8, 1 mM EDTA, 250 mM sucrose, and 10 pg/ml each of the protease inhibitors chymostatin, leupeptin, antipain, and pepstatin (allfrom Sigma). Cell extracts were centrifuged a t 30,000 X g for 30 min a t 4 "C, and s u p e r n a ~ n t s were used for assays. Cell extracts used in the immunoimmobilization assay (see below) wereprepared according to Ekanger and co-workers (41,42) DEAE-cellulose Chromatography-Fractionation of cAKI and cAKII was performed according to a method previously described (43). P h o s ~ h o t r a ~ f eActivity r~e of c A M P - d e ~ ~ Protein e n ~ KinaseCatalytic activity of cAK was assayed by phospho~lating thecAKspecific substrate kemptide (Peninsula Laboratories, Inc., Cat. no. 8650) using Y-[~'P]ATP(5000 mCi/mmol) in an assay mixture described by Roskoski et al. (44). Calculation of the C subunit concentration was based on the specific activity of homogenous bovine heart C subunit, (600 phosphates transferred/min/mol of C subunit). Protein Measurements-All protein measurement,swere done using ) on a method the Bio-Rad protein assay kit (no. 5 0 0 - ~ 6 based developed by Bradford (45). Cyclic AMP Binding and Immunoimmobilization Assays-Determination of specific cAMP binding of soluble R subunits was carried binding out according to Corbin et al. (2). Quantitation of [3H]~AMP to RI and RII subunits was done by immunoimmobilization assay as described by Ekanger and co-workers (41,42). Determinationof total R, RI, and RII subunit activities were based on two CAMP-bin~ng sites per monomer (2). S D ~ - P o l y ~ ~ Gel ~ ~ iElectrophoresis d e (SDS-PAGE)-SDSPAGE was performed as described by Laemmli (46). Samples were diluted in SDS sample buffer (62.5 mM Tris-HC1, pH 6.8, 2.3% SDS, 10% glycerol, 5% @-mercaptoethanol, 0.0001% bromphenol blue), boiled for 2 min and loaded onto slab gels consisting of a 4.5% stacking gel and a 7.5 or 10% separating gel (indicated in the figure legends). R ~ ~ i m m u n o ~ b e l(Western ing Blo~~-Westernblotting was performed according to a method described by Halbrugge et al. (47). Recognition of the T lymphocyte RI, was accomplished using a
monoclonal anti-human testis RI, antibody, diluted 1/500 as primary antibody.2 Determination of isomeric forms of RII (RII, and RHB) was accomplished using antisera (diluted 1/200) raised against synthetic peptides. The peptides used for immunization represent specific amino acid sequences within the €31, and RIIBproteins, respectively. Antisera raised against these peptides are highly specific for the human RII, and RI18.3 Photoaffinity Labeling of RSubunits by 8-Azido-pzP]-cAMPCovalent incorporation of the cAMP analog 8-a~ido-[~'P]-cAMP (ICN; specific activity, 64.8 Ci/mmol) into theregulatory (R) subunits of cAK was according to themethod of Walter and Greengard (48). P h o s p h o ~ ~ t i oan d D e p h o s p h o ~ ~ t i oofn RII Subunits-Phosphorylation and dephospho~lation of purified human testis RII subunits (50 ng) or human T lymphocyte RII subunits (in 200 pg of T lymphocyte cell homogenates) was carried out as described previously (38). Activation of T Lymphocytes-Purified T lymphocytes (5 X lo4 cells/well) were stimulated with anti-CD3 antibodies (37 ng/ml) in flat bottom 96-well microtiter plates (Falcon 3072, Becton Dickinson) and CD3 cell surface markers cross-linked using magnetic beads coated with sheep anti-mouse IgG (DYNAL, Dynabeads "450, Cat. no. 11002). Cell replication was measured, after incubating the cells for 72 h, during which [3Hjthymidine (20 pCi/well) was included for the last 12 h. All stimulations were done in triplicate, and thestandard deviation was consistently within 10% of the mean. Treatment of Actiuated T Lymphocytes with CAMP Analogs-Activated T lymphocytes were treated with different concentrations of various cAMP analogs (all from Sigma, except 8-piperidino-cAMP, which was a gift from Dr. K.L. Murray, Smith Kline & French Research, Ltd., Welwyn, United Kingdom). The cAMP analogs were added 30 min after cross-linking the CD3 cell surface markers as described above. The T lymphocytes were thereafter incubated under identical conditions as described above. The potency of each analog to inhibit cell proliferation by 50% (ICbo)was determined by measuring [3H]thymidine incorporation as a function of cAMP analog concentration. RESULTS
Identification of Subunits of cAK inHuman Peripheral Blood T Lymphocytes.-To determine which of the subunits of cAK are expressed in human T cells, we extracted RNA from highly purified human peripheral blood T lymphocytes. The cells were purified as described under "Materials and Methods" and were routinely monitored to be more than 98% pure T lymphocytes (resultsnotshown).Northernfilters containing T lymphocyte RNA were probed with cDNAs for all known human cAK subunits (RL, RIB,RIL, RHg, C,,Cg, and CT).As seen in Fig. 1,mRNAs for the subunits RI, (3.2 kb), RII, (7.0 kb), C, (1.7 kb), andCg (4.0 kb) were recognized by their corresponding cDNA probes, whereas mRNAs for RIB,RIIB,and C, were below levels of detection (not shown). To verify the presence of the respective R subunits at the protein level, we photoaffinity-labeled T lymphocyte cell extracts with 8-a~ido-[~~P]-cAMP. This revealed three proteins with apparent molecular masses of 49, 51, and 54 kDa (Fig. 2). In spite of the fact that mRNAs for only two of the R subunits (RI, and RII,)were detected by Northern blots, three proteinsincorporated specifically the8-a~ido-[~*P]cAMP after photoaffinity labeling. To investigate this, we fractionated T lymphocyte cell extracts by DEAE-cellulose chromatography in orderto separatecAKI from cAKII. From Fig, 3, A and B, both cAKI and cAKI1were identified as shown both by phosphotransferase activity and specific [3H] CAMP-binding. The 49-kDa protein was eluted bylow salt concentration (200 mM NaCl). The mRNA data, combined with the elution B. S. Skifhegg et at., unpublished data. 3B. Landmark, B. S. Skilhegg, T. Jahnsen,and manuscript in preparation.
V. Hansson,
T Lymphocytes
Cyclic AMP-dependent Protein Kinase Human in
RI,
RII,
C, C
A
P
15709
T .
7.5 kb
4.4 kb
28s
1
B
n
z
2.4 kb
D
1.4 kb
B
4
18s
FIG.1. Expression of mRNAs for cAK subunits in human peripheral blood T lymphocytes. Northern blot analysis of total RNA extracted from human peripheral blood T lymphocytes (6 X 10') is shown. RNA samples containing 20 eg were electrophoresed, hlotted onto nylon membranes, and hybridized with nick-translated cDNA probes for RI,,, RI,,, RII,,, RHti, C',, C,,, and C,. The figure demonstrates expression ofRI., (3.2 kb), RH,. (7.0 kb), C,, (1.7 kb), and C,, (4.0 kb). Thesize of the mRNAswere estimat,edby comparison to an RNA standard ranging from 1.4 t o 7.5 kb, as indicated by the nrrou1.s.
cAMP
-
+ f
94
C
Fraction number
!J E
r
43
? I
-.1112" 8
"
12
20
24
36
50
DEAE fracllons
FIG. 3. cAKI and cAKII in human peripheral blood T lymphocytes. Cell extracts from human peripheral blood T lymphocytes (10 mg of protein) were fractionated by DEAE-cellulose chromatography employing a linear salt gradient ranging from 0-400 mM NaCI (- - - ) . A , every second fraction was analyzed for phosphotransferase activity in the presence(W) or absence( 0 )of excess CAMP, revealing two peaks of specific cAK kinase activity. R, identical samples as analyzed in A were monitored for specific cAMP binding, revealing two distinct peaks corresponding to catalytic activity inA . C, based on the elution patterns seen in A and R , selected samples (DEAE fractions 8, 12, 20, from the first peak, and DEAE fractions 24, 36 and 50,from the second peak) were photoaffinity labeledwith 8a~ido-["~P]-cAMP andsuhjected toSDS-PAGE in 7.5% gels and autoradiography.
pattern fromDEAE-cellulose and size determination by photoaffinity labeling, indicated that the smaller R subunit most probably representsRI,, whereas the two largerproteins represented the RII subunits. Conclusive identification of the three proteins and explanationof why RII gave two bands on 4 43 SDS-PAGE was achieved by investigating the mobility shift of R subunits in T lymphocyte cell extracts treated by phosphorylation and dephosphorylation(see "Materials and Methods") and by Western blots employing subunit-specific antisera. Fig. 4 depicts that the49-kDa protein was recognized by the antiserum against RI,. The 51- and 54-kDa RIIs were both recognized by the RII, antiserum and revealed distinct Dye front phosphorylation/dephosphorylation. mobility shift upon Phosphorylation with M$+/ATP and the catalytic subunit of FIG. 2. Photoaffinity labeling of T lymphocyte cell extracts cAK was associated witha change in apparentmolecular mass with .azido-[:"P]-cAMP. T lymphocyte cell extract containing50 from 51 to 54 kDa, whereas dephosphorylation by alkaline p g of protein was photoaffinity labeled with 8-a~ido-[~'P]-cAMP in phosphatase shifted the relative electrophoretic mobility from the presence (+) or absence (-) of excessunlabeled CAMP. SDS51 kDa (Fig. 4). 54 to PAGE in 7.5% gels andautoradiographyindicatedthreeCAMPQuantitation and Intracellular Distribution of cAK in Hubinding proteins with apparent molecular masses of 49, 51, and 54 kDa. man PeripheralBlood T Lymphocytes-It has previously been I
67
Cyclic AMP-dependent Protein Kinase Human in
15710
shown thatcAKI and cAKIIdisplay unequal distribution and expressionin several cell types (49-51). Duetothis, we monitored both total activities and intracellular distribution of R and C subunits of cAK in highly purified T lymphocytes. This was accomplished by measuring total R, RI, RII, andC subunit activity in total cell homogenates, as well as in the cytosol and in the particulate fraction after centrifugationa t 100,000 X g. Cell homogenates and membrane fractionswere examined for R and C subunit activity after Triton X-100 treatment, whereas the cytosol was left untreated (see “Materials and Methods”). Total R subunit activity in the cell homogenate was determinedto be 0.91 pmol/mg of total protein (Table I). By immunoimmobilization assay, approximately 75% of the R activity was determined to be RI and the remaining 25% was determined to be RII, indicating a n RI/RII ratio of 3. Whereas most of the RI (more than75%)
Anti-RI,,
I
I
“ Human Testis
Human T cells
FIG. 4. Immunoblots of phosphorylated or dephosphorylated T lymphocyte cell extracts and purified human testis R subunits. T lymphocyte cell extract containing 200 pg of protein or 50 ng of purified human testis RIorRII were phosphorylated or dephosphorylated as described under “Materials and Methods.” All samples were resolved by SDS-PAGE in 10% gels and transferred to nitrocellulose filters. The filters were incubated with an anti-human testis RI,. monoclonal antibody, antisera made against peptides from unique amino acid domains of the human RII, or RII, proteins (see “Materials and Methods”). Immunoreactive proteins were visualized by ”51-labeledprotein A and autoradiography.
T Lymphocytes
was soluble, more than 90% of the RII was associated with the membranes (Table I). Thus, the RI/RII ratiocytosol in the was 26. The R/C ratio in the homogenate and cytosol was close to unity (1.1 and 0.95), whereas the R/C ratio in the membranes was somewhat higher(1.8). Effect of CAMP Analogs on T Lymphocyte ProliferationProliferating T lymphocytes, activated by cross-linking the CD3 cell surface markers,were incubated withvarious cAMP analogs a t concentrations from M to 5 X lo-’%M . The inhibitory potency of the various cAMP analogs was monitored by [”Hlthymidine incorporation (Fig. 5). We demonstrate that all the cAMP analogs tested inhibited [“]thymidine incorporation in a concentration-dependent manner. The concentration a t which each analog inhibited the [“Hlthymidine incorporation by 50% (ICm) was determined. This demonstrated that 8-chlorophenylthio-CAMP was the most potent analog (ICs0 = 30 p ~ followed ) by W,02-dibutyryl-cAMP (90 p M ) > 8-aminohexylamino-CAMP (150 p M ) > 8-S-methylcAMP (250 p M ) > W-monobutyryl-CAMP (300 p M ) > N6benzoyl-CAMP (350 p ~ >) 8-S-ethyl-CAMP (450 p ~ >) 8methylamino-CAMP (550 PM) > 8-bromo-CAMP(900 p M ) > 8-piperidino-CAMP(1100 p M ) . Use of site-selective analog pairs can to a certain extent distinguish between effects mediatedvia cAKIand cAKII(24, 25). We therefore repeated the experiment shown in Fig. 5, except that a subinhibitory concentrationof 8-methylaminocAMP (40pM, 7% of the ICs0 value for 8-methylamino-CAMP alone, Fig. 5) was included. 8-Methylamino-CAMP was used as a priming analog since it binds with comparable affinity to the B site of both RI and RII (Table 11). The results shown in Fig. 6 demonstrate a new and decreased ICs0 value (designated for all the W-analogs and 8-piperidino-cAMP, revealing a synergistic effect. Comparing the ratio 1C~,o/ICso* for each analog (designatedICs0 synergism) with theselectivity of the various cAMP analogs for site A of RI (KAIIKRI) (Table 11) revealed a positive correlation. The correlation is depictedin Fig. 7. A plot of the observed ICs0 synergism against the siteA selectivities forRII (KAr,/K~ll) showed little correlation (not shown). It should be noted that synergism with 8-methylamino-CAMPwas only noted for analogs with opposite (A site)selectivity for RI. Analogs with various degrees of B site selectivity failed to synergize. This is theoretically against the expectations from purified enzyme systems, where weakly B-selective analog complements somewhat a strongly B-selectiveanalog. However, in the intact cell, endogenous cAMP is present, offering a partner with neutral site selectivity for a strong B-selective analog. The break in the curveof Fig. 7 could therefore be expected. Proliferating T Lymphocytes Are Inhibitedby Selective Activation of cAKZ but Not cAKZZ-The data obtained (Fig. 7) revealed that cAMP analog complementation could be predicted quantitatively in intact cells according to data obtained
TABLE I Quantitation, intracellular distribution, and ratiosof CAMP-dependent protein kinaseR and C subunits in human peripheral blood T lymphocytes T lymphocyte cytosol and membrane fractions were prepared by Airfuge centrifugation (100,000 X g for 5 min) of total cell homogenates. Total R, RI, RII, and C subunit activities were measured and calculated as described under “Materials and Methods.” R and C subunit activities are given as mean values f SD of three seDarate T IvmDhocvte isolations. Specific activities
Fractions
RI
Homogenate Cytosol Membranes
Ratio
R 0.91 f 0.03 0.67 -C 0.01 0.26 f 0.02
pmollmg protein 0.68 f 0.01 0.23 f 0.02 0.530.021 f 0.02 f 0.005 0.16 2 0.03 0.22 f 0.04
0.84 f 0.03 26 0.720.95 f 0.04 0.8 0.14 f 0.03
1.1
1.8
3
15711
Cyclic AMP-dependent Protein Kinase in Human T Lymphocytes
a,
.-
70
E,
10
0 S
I-
-6
5
4 ~3
-6
5 4 -3
-6 -5 4 -3
~6 -5
4
3
-6 -5 4
3
cAMP analog concentration (log M) FIG. 5. Potency of different cAMP analogs to inhibit T lymphocyte proliferation. T lymphocytes were stimulated to proliferation by cross-linking the CD3 cell surface markers (see "Materials and Methods") and exposed for 72 h to varying concentrations M to 5 X M ) of the following cAMP analogs: W-benzoyl-CAMP ( W - B n z ) ,W-monobutyryl-CAMP ( W - m b ) ,W,02-dibutyryl-CAMP(W,@-db), 8chlorophenylthio-CAMP (8-CPT),8-aminohexylamino-CAMP (&AHA), 8-bromo-CAMP( 8 - B r ) ,8-piperidino-CAMP (&pip), 8-S-ethyl-CAMP ( 8 - S E ) ,8-S-methyl-CAMP( 8 - S M ) ,and 8-methylamino-CAMP(&MA). The potency of each analog to inhibit T lymphocyte proliferation is illustrated by ['Hlthymidine incorporation as a function of cAMP analogConcentration. The concentration(PM) of each analog that inhibited T lymphocyte proliferation by 50% is designated IC, (indicated by the arrows).
TABLEI1 Calculation of IC,, synergism and lists of site selectiuity for site A and B of RI and RII ICbosynergism for each analog is calculated as a ratio of their respective IC,, and IC,,* values determined in Fig. 5 and 6, respectively. The analogs (defined in the legend to Fig. 5) are listed according to decreasing ICbu synergism values, revealing a positive correlation with site selectivity of site A of RI. selectivity Site ( K ,values)" Selectivity for site A IC60 synergism Analogs (IC~~/IC~~*)
0.09
N6-mb 3.6 N6,@-dbh 8-pip W-Bnz 8-CPT 8-Br
6.7 6.0 4.0 3.3 1.6 1.1 1.1 1.0 1.2 1.0
8-SE 8-SM 8-AHA 8-MA
1.6
AI RII
BI
0.74 3.6 2.3 3.5 3.4 0.111.3 0.77 0.84 0.11 0.07
RI
0.09 0.065 0.18 1.7 1.0 2.2 2.9
3.3
AI1
0.046 4.1 0.05 0.034 0.028 0.021 0.026
BII
(KAIIKE,)
0.041 0.041 3.2 0.034 17 6.8 0.004 9.0 1.5 0.29 1.6
39 39 35 19 2 1.3 0.35 0.29 0.07 0.021
(KArlIKsn)
18 18 0.014 12 0.003 0.15
0.018 0.07 0.016
" Site selectivity (Ktvalues) are listed according to Dmkeland et al. (56). The available evidence suggests that dibutyryl-CAMP acts intracellularly through its metabolite monobutyryl-CAMP. Therefore, the data for monobutyryl-CAMP are relevant also for dibutyryl-CAMP.
a,
.E 20
0
g -
10
S
I-
02
d -5
4
-3
6 3
4
3
-6 -5 4 -3
-6 ~5 4
-3
~6 -5 4
~3
cAMP analog concentration (log M) FIG. 6. Concentration-dependent inhibition of T lymphocyte proliferation by different cAMP analogs in combination with a fixed concentration of 8-methylamino-CAMP.T lymphocytes were stimulated to proliferation by cross-linking the CD3 cell surface markers and exposed to two cAMP analogs. One analog (8-methylamino-CAMP) was present a t a subinhibitory concentration (40 PM, 7% of the ICbofor 8-methylamino-CAMP whenadded alone (Fig. 5)). The concentrationfor the other (complementary) analog was varied from 10"j M to 5 X IO-" M. The apparent IC,, for each analog in combination with 40 PM 8-methylamino-CAMP is designated ICsu*. The abbreviations for each cAMP analog, given in the respective figure panels, are explained in the legend to Fig. 5.
Cyclic AMP-dependent Protein Kinase
15712
in Human T Lymphocytes value for this analog) strongly synergized in the inhibition of cell proliferation. 8-Aminohexylamino-CAMPselects site B of both RI and RII, whereas 8-piperi~no-CAMP selects site A of RI and site B of RII (Table 11). This combination should therefore synergize in the activation of cAKI. In contrast, addition of 8-chlorophenylthio-CAMP at increasing concentrations, combined with a constant subinhibitory concentra(30 p ~ )showed , no such synergism tion of ~-benzoyl-CAMP in its inhibitory potency (Fig. 8B). This combination should show strong synergism for activation of cAKII (52). DISCUSSION
In the present study, we have investigated the levels and intracellular distribution of subunits of cAK and to what 0031 0125 05 2 8 32 extent cAKI or cAKII may mediate the inhibitory effects of Analog selecttvlty for slte A of RI (KAl/ K B , ) cAMP on human T lymphocyte proliferation. Human T lymphocytes express mRNAs for the R subunits FIG. 7. Efficiency of cAMP analogs to synergize with 8methylamino-CAMP in the inhibition of T lymphocyte prolif- RI, and RII, and theC subunits C, and C& The subunitsRI,, eration as a function of relative selectivity for site A of RI. RII,, and C, are previously shown to represent the ubiquitous Ten cAMP analogs (see the legend to Fig. 5 for the definitions of the forms, found in most tissues (3). In contrast, the Cu mRNA abbreviations) were tested for their potency to inhibit T lymphocyte reveals a more tissue- and cell-specific distribution and is proliferation alone or in combination with 40 pM 8-MA. In the figure, mainly located in the brain (12). Our results demonstrate yet IC6clsynergism for each analog is plotted as a function of analog selectivity for site A of RI (see Table 11). The straight line is drawn another cell type expressing the CBsubunit. Although mRNAs according to theleast squares method of linear regression (? = 0.891, for only two R subunits (RI, and RII,) were present in human starting with a point of coordinates ( l , l ) ,corresponding to the inter- T lymphocytes, photoaffinity labeling with 8 - a ~ i d o - [ ~ ~ P ] section of the dashed lines. cAMP revealed three distinct proteins with apparent molecular masses of 49,51, and 54 kDa, respectively. Further studies employing DEAE-cellulose chromatography and Western % A B analysis after phosphorylation/dephosphorylation clearly 40 I 1 demonstrated the 49-kDa protein as RI,, whereas the 51- and 54-kDa proteins represented the dephospho- and phosphoforms of RII,,, respectively. The size and properties of the RI, subunit and the phospho- and dephospho- forms of the RII, subunit in T lymphocytes are identical with those recently shown for human testisRI, and RII, (38). Our results by CAMP-binding and immunoimmobilization assay demonstrated that RI (RIJ was the major R subunit in human T lymphocytes (approximately 75%), whereas RII (RII,) constituted the remaining 25%. We also demonstrated "I 0 E 5 4 -3 0 -6 5 -9 S a striking difference in subcellular distribution (75% of RI cAMP analog concentration (log M) was cytosolic, whereas more than 90% of RII was associated FIG.8. Synergistic activationof cAKI and cAKII using site- with the membrane particles). These findings are in general selective cAMP analogs in intactT lymphocytes. Site-selective agreement with a previous study of a popdation of peripheral cAMP analogs complementing each other in activating either cAKI blood lymphocytes (53), which indicated that cAKII was or cAKII were tested for their ability to synergize in the inhibition of predominantly associated with the plasma membrane. Since T lymphocyte proliferation. A , the potency of 8-aminohexylamino- populations of peripheral leukocytes constitute only 55-70% cAMP alone (0)and in combination with a subinhibitory concentraT lymphocytes, these results probably imply that other cell tion (90 FM) of 8-piperidino-CAMP(A)was usedto selectively activate cAKI. Since 8-piperidino-CAMP selects site A of RI and 8-amino- populations of mononuclear white blood cells express the hexylamino-CAMP selects site B of both RI and RII (Table 11), they same cAK isozymes as T lymphocytes and with the same should only synergize in activating cAKI. B, the potency of 8-chlo- s u b c e l l u l ~localization. and in combination with N6-benzoylrophenyIthio-CAMPalone (e) The recent demonstration of specific RII-binding proteins cAMP (A) (30 p ~ is) used to selectively activate cAKII. Since 8- in certain tissues provides a mechanism for preferential aschlorophenylthio-CAMP selects site B of RII and N6-benzoyl-CAMP selects site A of RI and RII, the two analogs should complement each sociation of RII with subcellular structures (49-51). Specific other only in activating cAKII. See the legend to Fig. 5 for the binding of the R subunit to anchoring proteinswould tend to localize the interacting C subunit in a certain subcellular de~nitionsof the abbreviations. compartment and inclose proximity to relevant protein subwithpure cAK isozymes. T o supportthe suggestion that strates. The dramatic difference in subcellular localization of activation of cAKI but not of cAKII was sufficient to inhibit cAKI and cAKII indicates that the isomeric forms of cAK in DNA replication, we selected cAMP analogs complementing T lymphocytes may mediate different functions within the each other in synergistic activation of either cAKI or cAKII. cell. T o investigate if one or both of cAKI and cAKII participate Optimal analog concentrations were determined from the ininhibiting T lymphocyte replication, we employed siteexperiments shown in Figs. 5 and 6. Fig. 8A shows that proliferating T lymphocytes co-treated selective cAMP analog pairs on T lymphocytes activated to with 8-aminohexylamino-CAMPat increasing concentrations proliferate by cross-linking the CD3 cell surface markers. The and the complementary cAMP analog 8-piperidino-CAMPat theoretical basis for employing site-selective analog pairs in a constant s u b i n h i b i t o ~ concentration (90 pM, 7% of the ICso order to activate cAKI and cAKII, in uitro, has mainly been 0015
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OOH
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64
Cyclic AMP-dependent Protein Kinase in H u m a n T Lymphocytes
15713
worked out on purified isozymes from rabbit skeletal muscle cell replicationis a cAKI-mediated effect. Inhepatocytes, pairs and bovine cardiac muscle; both tissues contain the a-forms both cAKI- and cAKII-directed cAMP analogsynergized (56). Thus,itappearsthat of the cAK subunits (52, 54). In this respect, the human T ininhibitingDNAreplication CAMP-induced regulation of DNA replication may differ belymphocytes used in the present study represent an attractive model system, since they are not complicated by heterogeneity tween lymphoid cells and hepatocytes. A principal difference in their RI and RII subunits. Efficient activation of cAK betweenhepatocytesand lymphoid cells seemsto be the requires binding of cAMP to both sites (A and B) on the R difference in subcellular localization of cAKI and cAKII. In is almost subunit. By combining one cAMP analog that binds prefer- lymphoidcells, cAKIis soluble,whereascAKII exclusively membrane-bound. This is in contrast cAKI to and entially to one of the sites (e.g. site B of RI and RII) with cAKII in rat hepatocytes, whichreveal similar subcellular another analog that binds to the opposite site on isozyme one (e.g. site A of RI) and the same site (e.g. site B of RII) on the distribution; approximately 75% of both isozymes is soluble other isozyme, it is possible to achieve preferential activation (41). When taken together, our results and the findings of others of one cAK isozyme. (25, 57, 59) strongly support the concept that the isomeric Our initial experiments (Fig. 5), revealed that all cAMP analogs tested inhibitedT lymphocyte proliferation in a con- forms of cAK mayserve to mediate specific and different functions within a cell. centration-dependentmanner,butwithvaryingpotencies (IC," values varied from 30-1100 PM). The potencies of the REFERENCES variouscAMPanalogs differedmarkedly fromthe values 1. Kammer, G. M. (1988) Immunol. Today 9,222-228 obtained with purified cAK isozymes (52, 54, 55). However, 2. Corbin, J. D., Sugden, P. H., West, L., Flockhart, D. A,, Lincoln, T. M., and McCarthy, D. (1978) J. Biol. Chem. 2 6 3 , 3997-4003 when comparing the potencies to those required to obtain 3. Beebe, S., and Corbin, J. D. (1986) in The Enzymes (Boyer, P. D., and 50% cytolysis of a leukemia cell line (IPC-81) (25, 56) they Krebs, E. G., eds) Vol. 17, pp. 43-111, Academic Press, Inc., Orlando, FL 4. Hofman, F., Beavo, J. A,, Bechtel, P. J., and Krebs, E. G. (1975) J. Btol. were found to bevery similar, with the exception of the Chem. 2 6 0 , 7795-8001 analogs 8-methylamino-CAMP and 8-aminohexylamino5. Lee, D. C., Carmichael, D. F., Krebs, E. G., and McKnight, G. S. (1987) Proc. Natl. Acad. Sci. U. S. A. 80, 3608-3612 CAMP, which were 4- and 8-fold more potent in T lympho6. Titani, K., Sasagawa, T., Ericsson, L. H., Kumar, S., Smith, S. B., Krebs, cytes than in the IPC-81cells. Several factors may influence E. G., and Walsh, K. A. (1984) Biochemistry 23,4193-4199 7. Clegg, C. H., Cadd, G. G., and McKnight, G. S. (1988) Proc. Natl. Acad. the potencies of different cAMP analogs in intact cells, inSci. U. S. A. 85,3703-3707 cluding ability to penetrate the cell membrane, intracellular 8. Scott, J. D., Glaccum, M. B., Zoller, M. J., Uhler, M. D., Helfman, D. M., McKnight G. S., and Krebs, E. G. (1987) Proc. Natl. Acad. Sci. U. S. A. degradation, and affinityfor the binding sitesof RI and RII. 84,5192-5196 I n order to ensure that the cAMP analogs acted via cAK and, 9. Weldon, S. L., Mumby, M. C., and Taylor, S. S. (1985) J. Biol. Chem. 2 6 0 , fiAAn-fiAAR -_.-_" possibly, if activation of one isozyme was preferentially assoS., Ericsson, L. H.,Walsh,K. A., Fischer, E. H., and Titani, K. ciated with T lymphocyte cell replication, we tested cAMP 10. Shoji, (1983) Biochemistry 22,3702-3709 analog pairs expected to synergize in the activation of cAKI 11. Uhler, M. D., Carmiacbel, G. S., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 1300or cAKII, respectively. The cAKI-directed cAMP analog pair 1WA 8-aminohexylamino-CAMPand8-piperidino-CAMP clearly 12. Uhle;,-M. D., Chrivia, J. C., and McKnight, G. S. (1986) J. Biol. Chem. 2 6 1 , 15360-15363 synergized in the inhibition of T lymphocyte proliferation, 13. Beebe, S., 0yen, O., Sandberg, M., F r ~ y s a A., , Hansson, V., and Jahnsen, T. (1989) Mol. Endocrinol. 4,465-475 whereas the cAKII-directed pair, 8-chlorophenylthio-CAMP 14. Solberg, R., Tasken, K., Keiserud, A., and Jahnsen, T. (1991) Biochem. and W-benzoyl-CAMP did not. This strongly indicated that Biophys. Res. Commun. 176,166-172 15. Jahnsen, T., Hedin,L., Kidd, V. J., Beattie, W.G., Lohmann, S. M., Walter, cAKI mediates theinhibitory effects of cAMPinhuman T. Z., Schiltz, E., Browner, M., Goldman, D. U., Durica, J., Schultz, peripheral blood T lymphocytes. The fact that we did not Ratoosh, S. L., and Richards, J. S. (1986) J. Biol. Chem. 2 6 1 , 123521 12361 observe a cAKII synergism is probably due to the fact that 16. Sandberg, M., T a s k h , K., 0yen, O., Hansson, V., and Jahnsen, T. (1987) membrane localization of this isozyme will not make relevant Biochem. Biophys. Res. Commun. 149,939-945 O., Sandberg, M., Eskild, W., Levy, F. O., Knutsen, G., Beebe, S., substrates available for phosphorylation. Another possibility 17. 0yen, Hansson, V., and Jahnsen, T. (1988) Endocrinology 122,2658-2666 is thatcAKI and cAKII in this situation of extreme compart- 18. Dmkeland, S. 0. (1978) Biochem. Biophys. Res. Cornmun. 83,542-549 19. Rannels, S. R., and Corbin, J. D. (1980) J. Biol. Chem. 2 6 5 , 7085-7088 mentalization binds cAMP differentially and that cAKI is 20. D~skeland,S. O., and Qgreid, D. (1981) Int. J. Biochem. 1 3 , 1-19 activated at lower cAMP concentrations than cAKII. Fur- 21. Bramson, H. N., Kaiser, E. T., and Mildvan, A. S. (1984) CRC Crit. Reu. Biochem. 16,93-124 thermore, because cAKI is in excess of cAKII (approximately 22. 0greid, D., and D~skeland,S. 0. (1983) Biochemistry 2 2 , 1686-1696 3:l) in thesecells, possible synergistic effectsof CAMPanalogs 23. Robinson-Steiner, A. M., Beebe, S., Rannels, S. R., and Corbin,J. D. (1984) J. Biol. Chem. 2 5 9 , 10596-10605 on cAKII may to some extent be shaded by the basal effect 24. Beebe, S., Blackmore, P. F., Chrisman, T. D., and Corbin, J. D. (1988) of cAKI. However, Kammer et al. (57) recently demonstrated Methods Enzymol. 1 5 9 , 118-139 Lanotte, M., Riviere, J. B., Hermouet, S., Houge, G., Vintermyr, 0. K., 25. elevation of intracellular cAMP after stimulation of the T Gjertsen, B. T., and D~skeland,S. 0. (1991) J . Cell Physiol. 1 4 6 , 73-80 lymphocyte TCR-CD3 receptor complex. This was associated 26. Altman, A., and Coggeshall, K. M. (1990) Crit. Reu. Irnmunol. 10,347-391 27. Alexander, D. R., and Cantrell, D. A. (1989) Immunol. Today 10,200-205 with an enhanced capping of the CD3, CD4, and CD8 cell 28. Klausner, R. D., O'Shea, J. J., Luong, H., Ross, P., Bluestone, J. A,, and Samelson, L. E. (1987) J. Biol. Chem. 2 6 2 , 12654-12659 surface markers, without inhibitionof cell replication, which M., Samelson, L. E., and Klausner, R. D. (1987) J. Biol. Chem. 2 6 2 , results from cAMP generation upon prostaglandin E2 and 29. Patel, 5831-5835 30. Takayama, H., Tren, G., and Sitkovsky, M. V. (1988) J. Biol. Chem. 2 5 3 , forskolin stimulation (58, 59). ThisindicatesthatcAMP 2330-2336 formation in different subcellular compartments may have 31. Byus, C. V., Klimpel, G. R., Lucas, D. 0..and Russel, D. H. (1977) Nature 268,63-64 different biological effects, possibly due to preferential acti32. Beyurn, A. (1974) Tissue Antigens 4,269-273 vation of one cAK isozyme. 33. Smeland, E., Funderud, S., Ruud, F., Blomhoff, H. K., and Godal,T. (1985) Scand. J. Immunol. 2 1 , 205-214 Although the principle for synergistic activation of cAK 34. Melsom, H., Funderud, S., Lie, S. O.,andGodal, T. (1984) Scand. J . subtypes by cAMP analog pairs has been studied in vitro and Immunol. 3 3 , 27-34 in intact cells (24, 25, 56), very little information is available 35. Holte, H., Blomhoff, H.K., Beiske, K., Funderud, S., Torjesen, P., Gaudernack, G., Stokke, T., and Smeland, E. B. (1989) Eur. J. Immunol. 1 9 , about specific functions of cAMP that are mediated via one 1221-1225 T.,Vartdal, F., Davies, C., and Ugelstad, J. (1985) Scand. J . Immunol. specificR subunit.In a recentstudy,Lanotte et al. (25) 36. Lea, 22,207-216 demonstrated that DNA fragmentation and cell lysis (apop- 37. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutters, W. J. (1979) Biochemistry 18,5294-5299 tosis) can be induced exclusively by cAKI activation.Our 38. Skilhegg, B. S., Landmark, B., Foss, K. B., Lohmann, S. 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15714
Cyclic A ~ P - ~ e ~ nProtein ~ e n tKinasein Human T ~ y m p h o c ~ ~ e s
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50. Lohmann, S. M., DeCamilli, P., and Walter, U. (1988) Methods Enzyrnof. 159,183-193 51. Scott, J. D. (1991) ~ ~ r r n a c&o Tfter. ~ . 50, 123-145 eid, D. Ekanger R., Shuva, R. H. Miller, J. P., Sturm, P., Corbin, J. 52. and bmkeland, S. 0.(1985) Eu;, J. Biochern. 150,219-226 53. Chailin, D. D., Wedner, H.J., and Parker,C. W. (1980) in The B ~ l o g ~ u l Bars of Immunolo p .269-281 Raven Press NewYork 54. 0greid, D. Ekan er,%, ihuva, R. H., Miller, J. fi., and Dmkeland, S. 0. (1989 dur. J. hochem. 181, 19-31 Holloway, R., Rannels, S. R., and Corbin, J. D. (1984)J. Bid. 55. Beebe chek. 559 3539-3547 56. D~skeland,S.’O., B e , R. Brufand, T . , Vintermyr, 0. K., Jastorff, B., and Lanotte, M. (1991) in bell Sggnullmg: Enperzmental Strategtes, pp. 103114 57. Kammer, G. M., Boehm C. A., Rudol h, S. A,, and Scbultz, L. A. (1988) Proc. Natl. Acad. Sa. b. S. A. 85,782-796 58. Krause D. S and Deutsch C. (1991) J. Immunol. 146, 2285-2294 59. Kvand, A., J h d , M., andFredholm, B. B. (1991)Biochim. Biophys.Acta. 1093,178-183
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