Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1167-1171, March 1977 Genetics
Subunit interaction in cyclic AMP-dependent protein kinase of mutant lymphoma cells (somatic genetics/structural gene mutations/sodium thiocyanate)
JACOB HOCHMAN*, HENRY R. BOURNEtt§, PHILIP COFFINOt¶, PAUL A. INSEL#§, LESLIE KRASNYt*§, AND KENNETH L. MELMONt*§ * Department of Zoology, Hebrew University of Jerusalem, Israel; and Departments of t Medicine, * Pharmacology, and I Microbiology, and the § Cardiovascular Research Institute, University of California School of Medicine, San Francisco, California 94143
Communicated by N. 0. Kaplan, December 14, 1976
ABSTRACT We have previously selected and characterized mutant S49 mouse lymphoma cells that possess an adenosine 3':5'-cyclic monophosphate (cAMP)dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) with an increased apparent affinity constant (Ka) for activation by cAMP. The Ka lesion in one such mutant clone has been shown to result from a structural mutation involving the kinase holoenzyme's regulatory (R) subunit. The present report examines the interaction of R and catalytic (C) subunits of the kinases in extracts of the mutant cells and the normal "wild type" (WT) parental line. Subunit recombination experiments were performed, by using purified WVT and mutant R subunits, and C subunits purified from WT cells. As compared to VVT R subunits, only 14 as much mutant R subunit was required to reassociate with and suppress 50% of C subunit activity, at equilibrium. NaSCN activates cAMP-dependent kinase of both cell types by causing the holoenzyme to dissociate. In comparison with 'NT, a 2-fold higher concentration of NaSCN is required to maximally activate the kinase in mutant extracts. Both the reassociation result and the increased resistance of the mutant enzyme to a nonspecific dissociating agent strongly suggest that the mutant R subunit binds C subunit more tightly than does the 'NT R subunit. This interpretation raises the possibility that increased R-C subunit binding affinity in the mutant cell is responsible for the increased Ka for activation by cAMP of the mutant holoenzyme, and thus for the decreased potency of cAMP in regulating intact mutant cells.
Studies of mutant S49 lymphoma cells (1-6) have confirmed and extended the hypothesis (7) that the cellular actions of cyclic adenosine 3':5'-cyclic monophosphate (cAMP) are mediated by cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37). Mutant S49 clones may be completely or partially deficient in cAMP-dependent kinase activity, or may possess an enzyme with decreased apparent affinity for cAMP. In each case, the alteration in kinase activity directly corresponds with defective responsiveness of the intact mutant cell to cAMP actions, including enzyme induction, growth arrest in the G1 phase of the cell cycle, and cytolysis (5). In S49 (6) and other cells (8, 9), the cAMP-dependent kinase holoenzyme is composed of distinct regulatory (R) and catalytic (C) subunits. In the holoenzyme, R subunit inhibits C subunit activity; when cAMP binds to R the subunits dissociate, releasing catalytically active free C*: R C + cAMP cAMP R + C*. R-C subunit interactions in kinases from different tissues may also be regulated by distinct mechanisms that do not involve cAMP (10, 11). It is not known whether different mechanisms are important in modulating cAMP's control of cell functions. -
-
Mutations that affect the structure of an oligomeric protein may be used to help elucidate both the molecular basis of subunit interactions and their role in regulating intact cells. The phenotypes of one class of S49 cell mutants, i.e., those possessing a kinase with increased apparent Ka for cAMP, are produced by a structural mutation in the enzyme's R subunit, as shown by two lines of evidence: (i) experiments in which only the R subunit from the mutant cell was able to confer on a heterologously reconstituted holoenzyme the increased Ka and altered heat lability characteristic of the native mutant enzyme (6); (ii) altered electrophoretic mobility of mutant R subunits 11. In the present report, we have examined the interaction of R and C subunits of kinases derived from normal or "wild type" (WT) cells and from mutant cells expressing kinase activity with altered affinity for cAMP. The WT and mutant enzymes differ both in R-C subunit reassociation experiments and in their response to a chaotropic salt, NaSCN. The results suggest that a structural mutation in the R subunit peptide can significantly affect its ability to bind C subunits, either independently of or in addition to possible changes in R subunit affinity for binding cAMP. METHODS AND MATERIALS Cells. Mouse S49 lymphoma cells (12) were propagated in suspension culture (1). Each cell type used was originally isolated as a single clone in soft agar (3). The cell nomenclature and mode of selection are as follows (numbers in parentheses indicate the clone number, and references for the characterization and isolation of each are listed): WT (24.3.2) and kin.C (24.6.1) were derived from the same population of S49.1 cells, the latter in selective medium containing N6,02-dibutyryl cAMP and theophylline (3, 4). Clones kin.A (104.8.4) (5), kin.B (104.7.5) (5), and kin.D (6.3A)** were all selected for cAMP resistance from WT clone 24.3.2. Protein Kinase and [3H]cAMP Binding. Protein kinase activity was determined, as described (5), by measuring the transfer of 32p from [,y-32P]ATP (International Chemical Nuclear) to FIIB histone (Sigma) for a 5 min incubation at 300, in the presence or absence of 10,uM cAMP, a concentration which produced maximal activation in all the cell extracts examined. Binding of [3H]cAMP (New England Nuclear) to cell extracts was measured by the Millipore filter technique (13) after a 60 min incubation at 40 in 25 mM Tris-HCI, 50 mM sodium phosphate at pH 6.7, 2 mM dithiothreitol. The concentration of [3H]cAMP used (0.5 .M) produced maximal binding in all cell extracts. 1 R. A. Steinberg, P. H. O'Farrell, U. Friedrich, J. Hochman, and P. Coffino, manuscript in preparation. ** I. Lemaire and P. Coffino, manuscript in preparation.
Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; WT, wild type; TD buffer, 50 mM Tris-HCI at pH 7.5 and 4 mM dithiothreitol; R subunit, regulatory subunit; C subunit, catalytic subunit. 1167
1168
Proc. Nati. Acad. Sci. USA 74 (1977)
Genetics: Hochman et al.
Separation of R and C Kinase Subunits. As starting material for the separation of subunits from kin.A and WT cells, we used cAMP-dependent protein kinase partially purified as described (6) by salt elution initially from DEAE-cellulose column, then by Sephadex G-200 gel filtration; the second kinase peak from the G-200 column (6) was used in each case. This material was concentrated by ultrafiltration and applied to an affinity column of N6-(2-aminoethyl)-cAMP coupled to Sepharose 4B, as described by Dills et al. (14). R subunit (cAMP-binding) activity was eluted from the columns with 4 M guanidine hydrochloride (Schwarz/Mann Ultra Pure) (15) and dialyzed for 24 hr against 50mM Tris-HCI at pH 7.5 and 4 mM dithiothreitol (TD buffer). Column recovery of R subunit, from both WT and kin.A extracts, was 40-50%, as assessed by [3H]cAMP binding. C subunit activity in the effluent from the cAMP affinity column was further purified by application to a DEAE-cellulose column equilibrated with TD buffer; the bulk of the protein was retained by this second DEAE column, while 90-100% of C subunit activity was recovered in the column effluent. Protein in all the subunit preparations was below the limits of assay, and indicated at least a 250-fold purification with respect to cytosol fractions prepared from cell homogenates. R and C subunit preparations were both supplemented with 300 ug/ml of bovine serum albumin in order to prevent loss of activity. The R subunit preparations contained no detectable histone phosphotranserase activity, and conversely, [3H]cAMP binding could not be detected in C subunit preparations. cAMP produced no increase in phosphorylation of histone by the C subunit preparations (5). R-C Subunit Reassociation. Varying amounts of R subunit activity (measured by [3H]cAMP binding) from WT or kin.A cells were added to a constant amount of C subunit (obtained from WT cells) in a final volume of 0.05 ml TD buffer, and the mixtures were incubated at 40 for 18 hr. At this time kinase activity was measured in the presence or absence of cAMP. RESULTS Subunit Reassociation. The increased apparent Ka for cAMP of the kinase holoenzyme in kin.A cells, as compared with WT, appears to be due to a structural alteration of the kin.A R subunit (6). R-C subunit reassociation experiments, performed in the absence of cAMP, showed that the structural change in R subunits also alters its ability to recombine with and suppress C subunit activity (Fig. 1). At equilibrium (see below), high concentrations of both types of R subunit suppressed C subunit activity and rendered it sensitive to stimulation by cAMP; at low R subunit concentrations, however, the subunit prepared from kin.A cells was much more effective than WT (Fig. 1). Molar concentrations of C could not be measured (see Methods and Materials), and the stoichiometry of the reaction was not established (e.g., we do not know whether R subunit acted as a monomer or dimer under these conditions); therefore, it is not possible to assign affinity constants to the reassociation reactions. However, if we assume that the ability of the R preparations to bind [3H]cAMP (see Methods and Materials) is a valid measure of relative R concentrations, then a 6-fold higher concentration of WT R subunit is required to suppress 50% of C subunit activity. This suggests that the apparent affinity of kin.A R subunits for C subunit is greater than that of WT R subunit. After 18 hr (as in Fig. 1), reassociation of C subunits with both types of R subunits had reached equilibrium, as indicated by the fact that a further 10 hr incubation failed to increase the degree of R-C subunit recombination, at all R subunit concentrations tested (results not shown). An additional possibility
0
z 05 CD)
w
C
2 3 R BINDING ACTIVITY ADDED
4
(pmol/50 p1) FIG. 1. Recombination of WT C subunits with R subunits separated from cAMP-dependent kinase of WT (0) or kin.A (0) cells. A constant amount of C subunit activity was incubated for 18 hr at 40 with varying amounts of separated R subunits from the two cell types, as described in Methods and Materials; then histone phosphotransferase activity was measured in the presence or absence of 10 ,M cAMP. % Recombination = 100 X [A - BJ/C, where A and B represent the kinase activity (after recombination) measured in the presence and absence of cAMP, respectively, and C is the amount of C subunit activity added (400 pmol/min per tube). In replicate determinations, A and C did not differ by more than 10% for any of the points shown. Separation of R and C subunits and measurement of R subunit binding activity are described in Methods and Materials.
is that the different extents of recombination were caused by differential inactivation of R subunits during the reassociation period. However, when the experiment was repeated after a further 24 hr incubation of R subunits at 40, before allowing them to recombine with C subunits, the results were identical to those in Fig. 1. In the previous report (6) assigning the kin.A lesion to the kinase holoenzyme's R subunit, R and C subunit activity were separated by a different method (elution of C subunit by cAMP from a DEAE-cellulose ion-exchange column), not involving cAMP-Sepharose. Heterologous and homologous reconstitution experiments, using R and C subunits separated by the cAMPSepharose method, produced holoenzymes exactly similar to those described previously (6), i.e., whatever the source of the C subunit, recombination with R subunits prepared from WT cells produced a reconstituted holoenzyme with WT affinity for cAMP, and kin.A R subunits conferred on the reconstituted holoenzyme a Ka for cAMP characteristic of the native kin.A kinase (Table 1). Dissociation of Kinase Subunits by Thiocyanate. The results of the recombination experiments (Fig. 1), indicating different apparent affinities of WT and kin.A R subunits for C subunits, might have been due to differential susceptibility of the two R subunit peptides to denaturation during their preparation (e.g., by elution from cAMP-Sepharose with guanidine hydrochloride). Because we could not rule out this possibility, we sought another means of assessing the association between R and C subunits in the two kinases, without having to expose them to chromatography or to cAMP. Chaotropic agents, such as NaSCN have recently been shown (16) to dissociate free C subunit activity from the cAMP-dependent kinase of rabbit skeletal muscle. By using Sephadex G-100 gel filtration (Fig. 2), we found that 0.1 M NaSCN (Fig. 2B), but not 0.1 M NaCl (Fig. 2A) also dissociates cAMP-dependent kinase in cytosol of WT S49 cells. The kinase activity elutes in the column's excluded volume with sodium chloride, and is cAMP-dependent (Fig. 2A), but both NaSCN (Fig. 2B) and 10 AM cAMP (Fig. 2C) treatment of the cytosol caused kinase activity to be included in the gel and to elute in a peak corresponding to a molecular weight of 38,000 + 2,000, iden-
Proc. Natl. Acad. Sca. USA 74 (1977)
Genetics: Hochman et al.
1169
Table 1. Mutant cAMP-dependent kinases in S49 cells
Phenotype
(E4M)
NaSCN for maximal* activation (M)
WT kin.A kin.B kin.C kin.D
0.08 0.8 0.08 (no activation) 1.0
0.15 0.30 0.15 § 0.15
Ka for cAMP*
ty2 for thermal t inactivation (min) 10 36
Mutant
subunit*
11
R ?
3
R
?C§
A small amount of [3H]cAMP binding activity can be detected in kin.C cytosols (R. A. Steinberg and P. Coffino, manuscript in preparation). * Ka for cAMP and the concentration of NaSCN required for maximal activation were determined for the enzyme in cytosol (100,000 X g supernatant) fractions, as described in Methods and Materials and in the legend to Fig. 3. Ka values for all clones were identical to those published previously (5, 6) or to be described elsewhere**. t Determined by incubating cytosol extracts at 560, cooling to 40, and measuring kinase activity in the presence of 10 ,M cAMP, as described
(6).
Determined by reconstituting homologous and heterologous holoenzymes, as described (6). § Kin.C cytosols contain no detectable cAMP-stimulated kinase activity (5), and showed no response to NaSCN.
tical to that observed when purified C subunits prepared through the cAMP-Sepharose-DEAE-cellulose procedure are applied to the same column (Fig. 2D). This molecular weight is comparable to that reported for C subunits of kinases in other tissues (9). Kinase activity eluted in the presence of NaSCN is cAMP-independent, and implies that the C subunits have been separated from, and are thus no longer suppressed by, R subunits.
Accordingly, we tested the ability of various concentrations of NaSCN to activate kinase activity in cytosol preparations of WT and kin.A cells (Fig. 3). NaSCN increased kinase activity 300-
200-
A 200
-a~~~~~~~~10 EL
000
0
(measured in the absence of cAMP) in both cell extracts, but 2-fold higher concentrations of NaSCN were required for maximal activation in kin.A than in WT (0.3 M versus 0.15 M). Total kinase activity (measured in the presence of 10 ,uM cAMP) declined to a similar extent in both extracts at higher NaSCN concentrations, presumably because of an inhibitory effect of the salt on activity of free C subunits, also noted by other workers (16). The same pattern and difference between WT and kin.A enzymes were found when the NaSCN experiment of Fig. 3 was repeated with partially purified [through the Sephadex G-200 step (6)] cAMP-dependent kinase (not shown). This indicates that the effects of NaSCN are probably due to an interaction with the kinase itself, rather than with another cytosol protein that modulates its activity. The fact that higher concentrations of the dissociating agent, NaSCN, are required for maximal activation of kin.A than of WT cAMP-dependent kinase is consistent with the most direct interpretation of the recombination experiments, i.e., the affinity of kin.A R subunits for binding to (and suppression of) C subunits is greater than that of WT R subunits.
E
>300-
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E600 < 200-
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50
100
(D E400 _ 0
5 10
20
0
'U / %%~~~%
v
20
ELUTION VOLUME (ml)
FIG. 2. Dissociation, induced by NaSCN or cAMP, of S49 cell cAMP-dependent kinase activity. cAMP-dependent kinase in 0.4 ml of WT cytosol [100,000 x g (for 1 hr) supernatant fraction] (panels A-C) or purified C subunit activity (see Methods and Materials) (panel D) were applied to a Sephadex G-100 column (1.2 x 20 cm). Eluted kinase activity in 0.5 ml fractions was assayed in the presence (0-0) or absence (0-0) of 10MM cAMP. Prior to being put on the column, cytosol samples were incubated for 45 min at 40 with TD buffer containing 0.1 M NaCl, 0.1 M NaSCN, or 10MgM cAMP in 0.1 M NaCl (panels A, B, and C, respectively). In each case, the column was equilibrated with TD buffer containing the same concentration of salt or cAMP, and activity was eluted with the same solution. In panel D only TD buffer with 0.1 M NaCl was used.
Subunit interaction in cyclic AMP-dependent protein kinase of mutant lymphoma cells (somatic genetics/structural gene mutations/sodium thiocyanate)
JACOB HOCHMAN*, HENRY R. BOURNEtt§, PHILIP COFFINOt¶, PAUL A. INSEL#§, LESLIE KRASNYt*§, AND KENNETH L. MELMONt*§ * Department of Zoology, Hebrew University of Jerusalem, Israel; and Departments of t Medicine, * Pharmacology, and I Microbiology, and the § Cardiovascular Research Institute, University of California School of Medicine, San Francisco, California 94143
Communicated by N. 0. Kaplan, December 14, 1976
ABSTRACT We have previously selected and characterized mutant S49 mouse lymphoma cells that possess an adenosine 3':5'-cyclic monophosphate (cAMP)dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) with an increased apparent affinity constant (Ka) for activation by cAMP. The Ka lesion in one such mutant clone has been shown to result from a structural mutation involving the kinase holoenzyme's regulatory (R) subunit. The present report examines the interaction of R and catalytic (C) subunits of the kinases in extracts of the mutant cells and the normal "wild type" (WT) parental line. Subunit recombination experiments were performed, by using purified WVT and mutant R subunits, and C subunits purified from WT cells. As compared to VVT R subunits, only 14 as much mutant R subunit was required to reassociate with and suppress 50% of C subunit activity, at equilibrium. NaSCN activates cAMP-dependent kinase of both cell types by causing the holoenzyme to dissociate. In comparison with 'NT, a 2-fold higher concentration of NaSCN is required to maximally activate the kinase in mutant extracts. Both the reassociation result and the increased resistance of the mutant enzyme to a nonspecific dissociating agent strongly suggest that the mutant R subunit binds C subunit more tightly than does the 'NT R subunit. This interpretation raises the possibility that increased R-C subunit binding affinity in the mutant cell is responsible for the increased Ka for activation by cAMP of the mutant holoenzyme, and thus for the decreased potency of cAMP in regulating intact mutant cells.
Studies of mutant S49 lymphoma cells (1-6) have confirmed and extended the hypothesis (7) that the cellular actions of cyclic adenosine 3':5'-cyclic monophosphate (cAMP) are mediated by cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37). Mutant S49 clones may be completely or partially deficient in cAMP-dependent kinase activity, or may possess an enzyme with decreased apparent affinity for cAMP. In each case, the alteration in kinase activity directly corresponds with defective responsiveness of the intact mutant cell to cAMP actions, including enzyme induction, growth arrest in the G1 phase of the cell cycle, and cytolysis (5). In S49 (6) and other cells (8, 9), the cAMP-dependent kinase holoenzyme is composed of distinct regulatory (R) and catalytic (C) subunits. In the holoenzyme, R subunit inhibits C subunit activity; when cAMP binds to R the subunits dissociate, releasing catalytically active free C*: R C + cAMP cAMP R + C*. R-C subunit interactions in kinases from different tissues may also be regulated by distinct mechanisms that do not involve cAMP (10, 11). It is not known whether different mechanisms are important in modulating cAMP's control of cell functions. -
-
Mutations that affect the structure of an oligomeric protein may be used to help elucidate both the molecular basis of subunit interactions and their role in regulating intact cells. The phenotypes of one class of S49 cell mutants, i.e., those possessing a kinase with increased apparent Ka for cAMP, are produced by a structural mutation in the enzyme's R subunit, as shown by two lines of evidence: (i) experiments in which only the R subunit from the mutant cell was able to confer on a heterologously reconstituted holoenzyme the increased Ka and altered heat lability characteristic of the native mutant enzyme (6); (ii) altered electrophoretic mobility of mutant R subunits 11. In the present report, we have examined the interaction of R and C subunits of kinases derived from normal or "wild type" (WT) cells and from mutant cells expressing kinase activity with altered affinity for cAMP. The WT and mutant enzymes differ both in R-C subunit reassociation experiments and in their response to a chaotropic salt, NaSCN. The results suggest that a structural mutation in the R subunit peptide can significantly affect its ability to bind C subunits, either independently of or in addition to possible changes in R subunit affinity for binding cAMP. METHODS AND MATERIALS Cells. Mouse S49 lymphoma cells (12) were propagated in suspension culture (1). Each cell type used was originally isolated as a single clone in soft agar (3). The cell nomenclature and mode of selection are as follows (numbers in parentheses indicate the clone number, and references for the characterization and isolation of each are listed): WT (24.3.2) and kin.C (24.6.1) were derived from the same population of S49.1 cells, the latter in selective medium containing N6,02-dibutyryl cAMP and theophylline (3, 4). Clones kin.A (104.8.4) (5), kin.B (104.7.5) (5), and kin.D (6.3A)** were all selected for cAMP resistance from WT clone 24.3.2. Protein Kinase and [3H]cAMP Binding. Protein kinase activity was determined, as described (5), by measuring the transfer of 32p from [,y-32P]ATP (International Chemical Nuclear) to FIIB histone (Sigma) for a 5 min incubation at 300, in the presence or absence of 10,uM cAMP, a concentration which produced maximal activation in all the cell extracts examined. Binding of [3H]cAMP (New England Nuclear) to cell extracts was measured by the Millipore filter technique (13) after a 60 min incubation at 40 in 25 mM Tris-HCI, 50 mM sodium phosphate at pH 6.7, 2 mM dithiothreitol. The concentration of [3H]cAMP used (0.5 .M) produced maximal binding in all cell extracts. 1 R. A. Steinberg, P. H. O'Farrell, U. Friedrich, J. Hochman, and P. Coffino, manuscript in preparation. ** I. Lemaire and P. Coffino, manuscript in preparation.
Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; WT, wild type; TD buffer, 50 mM Tris-HCI at pH 7.5 and 4 mM dithiothreitol; R subunit, regulatory subunit; C subunit, catalytic subunit. 1167
1168
Proc. Nati. Acad. Sci. USA 74 (1977)
Genetics: Hochman et al.
Separation of R and C Kinase Subunits. As starting material for the separation of subunits from kin.A and WT cells, we used cAMP-dependent protein kinase partially purified as described (6) by salt elution initially from DEAE-cellulose column, then by Sephadex G-200 gel filtration; the second kinase peak from the G-200 column (6) was used in each case. This material was concentrated by ultrafiltration and applied to an affinity column of N6-(2-aminoethyl)-cAMP coupled to Sepharose 4B, as described by Dills et al. (14). R subunit (cAMP-binding) activity was eluted from the columns with 4 M guanidine hydrochloride (Schwarz/Mann Ultra Pure) (15) and dialyzed for 24 hr against 50mM Tris-HCI at pH 7.5 and 4 mM dithiothreitol (TD buffer). Column recovery of R subunit, from both WT and kin.A extracts, was 40-50%, as assessed by [3H]cAMP binding. C subunit activity in the effluent from the cAMP affinity column was further purified by application to a DEAE-cellulose column equilibrated with TD buffer; the bulk of the protein was retained by this second DEAE column, while 90-100% of C subunit activity was recovered in the column effluent. Protein in all the subunit preparations was below the limits of assay, and indicated at least a 250-fold purification with respect to cytosol fractions prepared from cell homogenates. R and C subunit preparations were both supplemented with 300 ug/ml of bovine serum albumin in order to prevent loss of activity. The R subunit preparations contained no detectable histone phosphotranserase activity, and conversely, [3H]cAMP binding could not be detected in C subunit preparations. cAMP produced no increase in phosphorylation of histone by the C subunit preparations (5). R-C Subunit Reassociation. Varying amounts of R subunit activity (measured by [3H]cAMP binding) from WT or kin.A cells were added to a constant amount of C subunit (obtained from WT cells) in a final volume of 0.05 ml TD buffer, and the mixtures were incubated at 40 for 18 hr. At this time kinase activity was measured in the presence or absence of cAMP. RESULTS Subunit Reassociation. The increased apparent Ka for cAMP of the kinase holoenzyme in kin.A cells, as compared with WT, appears to be due to a structural alteration of the kin.A R subunit (6). R-C subunit reassociation experiments, performed in the absence of cAMP, showed that the structural change in R subunits also alters its ability to recombine with and suppress C subunit activity (Fig. 1). At equilibrium (see below), high concentrations of both types of R subunit suppressed C subunit activity and rendered it sensitive to stimulation by cAMP; at low R subunit concentrations, however, the subunit prepared from kin.A cells was much more effective than WT (Fig. 1). Molar concentrations of C could not be measured (see Methods and Materials), and the stoichiometry of the reaction was not established (e.g., we do not know whether R subunit acted as a monomer or dimer under these conditions); therefore, it is not possible to assign affinity constants to the reassociation reactions. However, if we assume that the ability of the R preparations to bind [3H]cAMP (see Methods and Materials) is a valid measure of relative R concentrations, then a 6-fold higher concentration of WT R subunit is required to suppress 50% of C subunit activity. This suggests that the apparent affinity of kin.A R subunits for C subunit is greater than that of WT R subunit. After 18 hr (as in Fig. 1), reassociation of C subunits with both types of R subunits had reached equilibrium, as indicated by the fact that a further 10 hr incubation failed to increase the degree of R-C subunit recombination, at all R subunit concentrations tested (results not shown). An additional possibility
0
z 05 CD)
w
C
2 3 R BINDING ACTIVITY ADDED
4
(pmol/50 p1) FIG. 1. Recombination of WT C subunits with R subunits separated from cAMP-dependent kinase of WT (0) or kin.A (0) cells. A constant amount of C subunit activity was incubated for 18 hr at 40 with varying amounts of separated R subunits from the two cell types, as described in Methods and Materials; then histone phosphotransferase activity was measured in the presence or absence of 10 ,M cAMP. % Recombination = 100 X [A - BJ/C, where A and B represent the kinase activity (after recombination) measured in the presence and absence of cAMP, respectively, and C is the amount of C subunit activity added (400 pmol/min per tube). In replicate determinations, A and C did not differ by more than 10% for any of the points shown. Separation of R and C subunits and measurement of R subunit binding activity are described in Methods and Materials.
is that the different extents of recombination were caused by differential inactivation of R subunits during the reassociation period. However, when the experiment was repeated after a further 24 hr incubation of R subunits at 40, before allowing them to recombine with C subunits, the results were identical to those in Fig. 1. In the previous report (6) assigning the kin.A lesion to the kinase holoenzyme's R subunit, R and C subunit activity were separated by a different method (elution of C subunit by cAMP from a DEAE-cellulose ion-exchange column), not involving cAMP-Sepharose. Heterologous and homologous reconstitution experiments, using R and C subunits separated by the cAMPSepharose method, produced holoenzymes exactly similar to those described previously (6), i.e., whatever the source of the C subunit, recombination with R subunits prepared from WT cells produced a reconstituted holoenzyme with WT affinity for cAMP, and kin.A R subunits conferred on the reconstituted holoenzyme a Ka for cAMP characteristic of the native kin.A kinase (Table 1). Dissociation of Kinase Subunits by Thiocyanate. The results of the recombination experiments (Fig. 1), indicating different apparent affinities of WT and kin.A R subunits for C subunits, might have been due to differential susceptibility of the two R subunit peptides to denaturation during their preparation (e.g., by elution from cAMP-Sepharose with guanidine hydrochloride). Because we could not rule out this possibility, we sought another means of assessing the association between R and C subunits in the two kinases, without having to expose them to chromatography or to cAMP. Chaotropic agents, such as NaSCN have recently been shown (16) to dissociate free C subunit activity from the cAMP-dependent kinase of rabbit skeletal muscle. By using Sephadex G-100 gel filtration (Fig. 2), we found that 0.1 M NaSCN (Fig. 2B), but not 0.1 M NaCl (Fig. 2A) also dissociates cAMP-dependent kinase in cytosol of WT S49 cells. The kinase activity elutes in the column's excluded volume with sodium chloride, and is cAMP-dependent (Fig. 2A), but both NaSCN (Fig. 2B) and 10 AM cAMP (Fig. 2C) treatment of the cytosol caused kinase activity to be included in the gel and to elute in a peak corresponding to a molecular weight of 38,000 + 2,000, iden-
Proc. Natl. Acad. Sca. USA 74 (1977)
Genetics: Hochman et al.
1169
Table 1. Mutant cAMP-dependent kinases in S49 cells
Phenotype
(E4M)
NaSCN for maximal* activation (M)
WT kin.A kin.B kin.C kin.D
0.08 0.8 0.08 (no activation) 1.0
0.15 0.30 0.15 § 0.15
Ka for cAMP*
ty2 for thermal t inactivation (min) 10 36
Mutant
subunit*
11
R ?
3
R
?C§
A small amount of [3H]cAMP binding activity can be detected in kin.C cytosols (R. A. Steinberg and P. Coffino, manuscript in preparation). * Ka for cAMP and the concentration of NaSCN required for maximal activation were determined for the enzyme in cytosol (100,000 X g supernatant) fractions, as described in Methods and Materials and in the legend to Fig. 3. Ka values for all clones were identical to those published previously (5, 6) or to be described elsewhere**. t Determined by incubating cytosol extracts at 560, cooling to 40, and measuring kinase activity in the presence of 10 ,M cAMP, as described
(6).
Determined by reconstituting homologous and heterologous holoenzymes, as described (6). § Kin.C cytosols contain no detectable cAMP-stimulated kinase activity (5), and showed no response to NaSCN.
tical to that observed when purified C subunits prepared through the cAMP-Sepharose-DEAE-cellulose procedure are applied to the same column (Fig. 2D). This molecular weight is comparable to that reported for C subunits of kinases in other tissues (9). Kinase activity eluted in the presence of NaSCN is cAMP-independent, and implies that the C subunits have been separated from, and are thus no longer suppressed by, R subunits.
Accordingly, we tested the ability of various concentrations of NaSCN to activate kinase activity in cytosol preparations of WT and kin.A cells (Fig. 3). NaSCN increased kinase activity 300-
200-
A 200
-a~~~~~~~~10 EL
000
0
(measured in the absence of cAMP) in both cell extracts, but 2-fold higher concentrations of NaSCN were required for maximal activation in kin.A than in WT (0.3 M versus 0.15 M). Total kinase activity (measured in the presence of 10 ,uM cAMP) declined to a similar extent in both extracts at higher NaSCN concentrations, presumably because of an inhibitory effect of the salt on activity of free C subunits, also noted by other workers (16). The same pattern and difference between WT and kin.A enzymes were found when the NaSCN experiment of Fig. 3 was repeated with partially purified [through the Sephadex G-200 step (6)] cAMP-dependent kinase (not shown). This indicates that the effects of NaSCN are probably due to an interaction with the kinase itself, rather than with another cytosol protein that modulates its activity. The fact that higher concentrations of the dissociating agent, NaSCN, are required for maximal activation of kin.A than of WT cAMP-dependent kinase is consistent with the most direct interpretation of the recombination experiments, i.e., the affinity of kin.A R subunits for binding to (and suppression of) C subunits is greater than that of WT R subunits.
E
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50
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5 10
20
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20
ELUTION VOLUME (ml)
FIG. 2. Dissociation, induced by NaSCN or cAMP, of S49 cell cAMP-dependent kinase activity. cAMP-dependent kinase in 0.4 ml of WT cytosol [100,000 x g (for 1 hr) supernatant fraction] (panels A-C) or purified C subunit activity (see Methods and Materials) (panel D) were applied to a Sephadex G-100 column (1.2 x 20 cm). Eluted kinase activity in 0.5 ml fractions was assayed in the presence (0-0) or absence (0-0) of 10MM cAMP. Prior to being put on the column, cytosol samples were incubated for 45 min at 40 with TD buffer containing 0.1 M NaCl, 0.1 M NaSCN, or 10MgM cAMP in 0.1 M NaCl (panels A, B, and C, respectively). In each case, the column was equilibrated with TD buffer containing the same concentration of salt or cAMP, and activity was eluted with the same solution. In panel D only TD buffer with 0.1 M NaCl was used.
