A Cytosolic Cyclic AMP-dependent Protein Kinase in Dictyostelium discoideum

A CAMP-dependent protein kinase was isolated and partially purified from Dictyostelium discoideum. The cytosolic holoenzyme has an apparent M, = 160,000-180,000; its activity was stimulated significantly by cAMP when Kemptide served as substrate. The enzyme was dissociated and the regulatory subunit purified by affinity chromatography on 8-aminoethylamino-CAMP. Only one type of regulatory subunit was found; it has an apparent M, = 41,000 and is a substrate for the in vitro phosphorylation by the homologous cata- lytic subunit and by purified bovine catalytic subunit. Antibody against the regulatory subunit was prepared. The D. discoideum catalytic subunit was separated from CAMP-independent protein kinase by chromato- focusing. The apparent molecular weight of the catalytic subunit of the D. discoideum CAMP-dependent protein kinase is 33,000 and its PI is 6.4. The enzyme catalyzed the phosphorylation of bovine RIr but not of RI regulatory subunit and was inhibited by high con- centrations of the inhibitor of mammalian CAMP-de-pendent protein kinase. The evolution of the functional domains of CAMP-dependent protein kinases is dis-cussed on the basis of a comparison of the analogous D. discoideum and vertebrate enzymes.

A regulatory role of cAMP in both prokaryotes and eukaryotes is established. There is evidence that in the prokaryote Escherichia coli, for example, cAMP exerts its functions, probably exclusively, by the control of the synthesis of certain proteins. The cyclic nucleotide modulates the formation of these proteins by interaction with the CAMP-binding protein (referred to as catabolite gene activator protein, or CAP) which, when complexed with CAMP, has high affinity for nucleotide sequences which form part of the promoters of genes controlled in their transcription by CAMP. The binding of the CAP-CAMP complex to the promoter DNA facilitates the transcription of the cognate genes (1). In eukaryotes cAMP affects both the syntheses and the activities of a variety of proteins. There is evidence that cAMP controls the for-*This research was supported by Grant ACS-BC-375 from the American Cancer Society. A preliminary report of these findings was presented at the European Molecular Biology Organization-Max-Planck-Gesellschaft Workshop in Tutzing, September 1981. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
7 Ida and Cecil Green Investigator in Developmental Biochemistry. mation of at least several eukaryotic proteins at the level of transcription (2-5). The mechanism of this control, however, is not known; e.g. it is not clear whether the effect of cAMP is mediated by a CAP-like binding protein or by a CAMPdependent protein kinase (the two possibilities are not necessarily mutually exclusive), or by yet some other mechanism. Cyclic AMP plays a central role in the development of the cellular slime mold, Dictyostelium discoideum, and this simple eukaryote has become a favorite model for the study of the mechanism of action of cAMP in eukaryotes. The organism synthesizes and releases cAMP into its environment when deprived of nutrients. The extracellular cAMP then acts as a chemotactic, morphogenetic agent and causes the individual, vegetative amoebae to form aggregates which eventually give rise to fruiting bodies. The latter consist of moribund stalk cells and spores with the potential to germinate into new vegetative amoebae. Cyclic AMP plays a dual role; the situation is unusual insofar as the cyclic nucleotide acts as intracellular second messenger for the intercellular signal or "hormone'' which is also CAMP; the two functions of cAMP are linked via the stimulation of the membranal adenylate cyclase by extracellular CAMP. What lends particular interest to the study of the role of cAMP in D. discoideum is the fact that the cyclic nucleotide appears to influence development of the slime mold, presumably by its regulatory role in protein synthesis. Evidence for an effect of cAMP on development comes from several sources. Thus, for example, exogenous cAMP brings about precocious development (6,7); in certain strains and under certain conditions exogenous cAMP leads to cellular differentiation into either stalk cells or spores even in the absence of morphogenesis, i.e. under conditions where a fruiting body is not formed (8)(9)(10). That these effects are indeed mediated by an impact, direct or indirect, of cAMP on transcription, is suggested by experiments in which multicellular slugs of D. discoideum were disaggregated into individual amoebae and where the persistence of certain slug-specific species of mRNA depended on the addition of cAMP to the suspension of amoebae; cAMP seemed to have an effect on both the synthesis and the stability of different stage-specific mRNAs (11,12). In other experiments exogenous cAMP reduced the rate of transcription of the discoidin I gene (2).
In view of what has been stated earlier about possible mechanisms by which cAMP might regulate protein synthesis, it becomes relevant to examine D. discoideum for the occurrence of CAMP-binding proteins and of CAMP-dependent protein kinases. The fact that D. discoideum is a very primitive eukaryote, which appears to derive from the deepest known eukaryotic branch of the evolutionary tree (13), lends added interest to the problem.
The occurrence of CAMP-binding proteins in D. discoideum has been observed in a number of laboratories (14-19

EXPERIMENTAL PROCEDURES
Strain and Conditions of Growth and Development-D. discoideum, strain AX3/RC3 (obtained from D. Soll, University of Iowa), was grown axenically at 22 "C on Medium HL5 (26), supplemented with 50 mM glucose, to a density of approximately lo' amoebae/ml. Amoebae were harvested by sedimentation and plated on PDF (27) agar (50 mM potassium phosphate, pH 6.5; 20 mM potassium chloride; 2.5 mM magnesium chloride; 2.0% Bacto agar) at approximately lo9 amoebae/l5 cm diameter Petri dish. For a typical purification procedure 1-2 x 10" amoebae, harvested from 14 liters of culture, were employed. Development was allowed to proceed for 18 h at 22 "C; at this time the "finger" stage of development (28) had been reached.
Partial Purification of CAMP-dependent Protein Kinase-All procedures were carried out at 0-4 "C. The developing amoebae were resuspended in lysis buffer (10 mM MOPS,' pH 7.2; 5 mM sodium chloride; 2 mM EDTA 4 mM mercaptoethanol; 1.0 mM PMSF; 20% sucrose), collected by sedimentation, and resuspended to a density of 5 X 10' amoebae/ml. The amoebae were then disrupted, without prior freezing, by sonication in a sonicator cell disruptor (Heath Systems Ultrasonics). Debris was removed by centrifugation for 10 min at 10,000 X g. The supernatant fraction was clarified further by a 1-h centrifugation at 40,000 X g and glycerol was added to a final concentration of 10%. The pH of the 40,000 x g supernatant fraction, typically containing about 5 g of amoeba1 protein, was adjusted to 7.2, and the material was adsorbed to a DE52 column (5 X 12 cm). After washing with approximately 3 bed volumes of buffer (5 mM MOPS, pH 7.2; 5 mM sodium chloride; 1 mM EDTA 2 mM mercaptoethanol; 0.5 mM PMSF; 10% glycerol) the column was eluted with a 5-500 mM gradient of sodium chloride; the total gradient volume was 1.75 liters. Fractions containing CAMP-dependent protein kinase activity were pooled and concentrated to about one-tenth of the original volume in an Amicon ultracentrifugation cell (YM10 Diaflo filter). The concentrate was applied to a Sephadex G-150 column (2.5 X 90 cm) previously equilibrated with Buffer A (5 mM MOPS, pH 7.0, containing 25 mM sodium chloride, 0.1 mM EDTA, 2 mM mercaptoethanol, 0.5 mM PMSF, and 10% glycerol); 4-ml fractions were collected and assayed for protein kinase activity. Fractions containing the CAMP-dependent enzyme were pooled and stored at -80 "C.
Preparation kinase in the pooled DE52 fractions was dissociated into its regulatory and catalytic subunits by passage through a column of 8-aminoethylamino-CAMP-Sepharose according to the procedure of Dills et al. (29). The high affinity CAMP-binding protein, assayed at pH 7, was retained quantitatively on the column whereas all the protein kinase activity, no longer CAMP-dependent, passed through the column. The column was washed successively with Buffer A, Buffer A containing 1 M NaC1, and again with Buffer A. The regulatory subunit, i.e. the high affinity CAMP-binding protein, was then eluted by incubation of the column at 37 "C for 30 min in Buffer A containing 1 mM cAMP followed by washing at 37 "C with the same buffer. No regulatory subunit was recovered by washing with 5'-AMP prior to the CAMP wash and no additional subunit was released from the column by washing with 30 mM cAMP or by 6 M urea after washing with 1 mM CAMP. Identical results were obtained when a column of 6-aminoethyl-CAMP-Sepharose was employed. The flow-through fraction, containing the protein kinase activity, was dialyzed against 20 mM MOPS buffer, pH 6.5, containing 2 mM EDTA, 4 mM mercaptoethanol, 1 mM PMSF, and 10% glycerol and passed through a 3-ml CM52 column pre-equilibrated with the same buffer. After washing, the column was eluted with a 0-500 mM sodium chloride gradient; 4.5-ml fractions were collected and assayed for protein kinase activity. Those fractions which contained activity were pooled and stored at -80 "C after the pH had been adjusted to pH 7.2. This protein kinase preparation was insensitive to stimulation by cAMP and sensitive to the mammalian inhibitor, specific for CAMP-dependent protein kinase (30); the activity of the preparation was inhibited also by the homologous free regulatory subunit obtained from the DE52 column.
Sucrose Density Gradient Centrifugation-Sucrose density gradients were done according to the method of Martin and Ames (31); linear 13-ml gradients of 5-17% sucrose in Buffer A were centrifuged for 20 h at 3 "C in a Beckman SW 41 rotor; 0.3-ml fractions were collected from the bottom of the tubes with a peristaltic pump. Gel Electrophoresis-Gel electrophoresis was performed as described earlier (32) according to the procedures of Laemmli (33) for one-dimensional gels and of O'Farrell (34) for two-dimensional gels. Proteins were stained either with Coomassie brilliant blue or silver (35). Autoradiography was done as described earlier (32). Antibody to Regulatory Subunit-The fraction eluted from the 8aminoethylamino-CAMP-Sepharose column by 1 mM cAMP was lyophilized, redissolved in SDS electrophoresis buffer (32), and submitted to electrophoresis on a preparative 10% acrylamide gel. The bands were visualized by soaking the gel in 4 M sodium acetate according to the procedure of Higgins and Dahmus (36). The major band, corresponding to a protein of molecular weight 41,000, was excised, rinsed with water, and minced by passage through a 16-gauge needle. After elution over a period of 2 days into three changes of 0.1% SDS, containing 0.01 M ammonium bicarbonate, the material, i.e. the purified regulatory subunit, was lyophilized and used for injection. Free regulatory subunit, obtained from the DE52 column, was also purified by cAMP affinity chromatography and preparative gel electrophoresis and used for immunization. Rabbits were given a primary injection into the popliteal gland of 50 pg of regulatory subunit suspended in complete Freund's adjuvant and subsequent intramuscular injections (3-4-week intervals) of 50 pg of regulatory subunit suspended in incomplete adjuvant. The serum was used without further purification after removal of the clot.
For the monitoring of CAMP-binding proteins, reactive with the antibody, extracts of D. discoideurn were photoaffinity labeled with 8N3-[32P]cAMP at a final concentration of 200 nM and incubated with the antiserum at 4 'C for 30 min. The antibody-antigen complex was then precipitated with heat-killed, formalized Staphylococcus aureus, Cowan strain, and washed according to the procedure of Erikson et al. (37). The staphylococci were prepared by the procedure of Kessler (38). The bound antigen was released by boiling in SDS electrophoresis buffer and then subjected to one-dimensional gel electrophoresis and autoradiography.
Assays-Cyclic AMP-binding activity was determined as described earlier (15); I3H]cAMP was employed at a concentration of 10 nM and at a pH of 7. Protein kinase activity was assayed with 100 MM Kemptide as substrate; in addition to Kemptide, the reaction mixture by guest on March 23, 2020 http://www.jbc.org/ Downloaded from contained, in a final volume of 0.1 ml, 25 mM MOPS buffer, pH 7.0, 4 mM magnesium chloride, 2 mM dithiothreitol, 5 mM sodium fluoride, 100 p M [y-32P]ATP at 100-500 cpm/pmol, and either 1 or 5 p~ CAMP, when present. Incubation was at 30 "C for 10-15 min. The reaction was terminated by the addition of 1 ml of ice-cold 30% acetic acid. The phosphorylated Kemptide was separated from the [y-"P] ATP as described by Kemp (39) and the radioactivity was determined by liquid scintillation counting. At an enzyme activity of 1 pmol/min, the reaction was rectilinear for at least 60 min. Proteins were quantitated by the method of Lowry et al. (40); bovine serum albumin served as standard.
Materials-Kemptide was purchased from both Peninsular Research Laboratories and Boehringer Mannheim. Worthington supplied histone H2B; Sigma furnished mixed histones, histone H1, acasein, and beef heart CAMP-dependent protein kinase; New England Nuclear supplied [methyl-"C]methylated y-globulin and [methyl-14C] methylated ovalbumin. Bacto agar and Freund's adjuvants were Difco products, Sephadex G-150 and the chromatofocusing kit were provided by Pharmacia, the CAMP-affinity resins by P-L Biochemicals and the DEAE-cellulose (DE52) and carboxymethylcellulose (CM52) by Whatman. [y-32P]ATP was either prepared by the method of Johnson and Walseth (41) or purchased from New England Nuclear. 8N3-[32P]cAMP was given to us by Boyd Haley, University of Wyoming, or purchased from ICN. Other chemicals were reagent grade or better.

Partial Purification of CAMP-dependent Protein Kinase-
When crude extracts of D. discoideum were centrifuged at 40,000 x g and the supernatant fractions were tested for protein kinase activity, such activity was found; however, it was stimulated only minimally (20-50%) and variably by cAMP (data not shown). Consistent stimulation of protein kinase activity was detected after the extract was passed through a DE52 column. The peak of CAMP-dependent protein kinase activity was eluted at a concentration of 30-50 mM NaCl. Inspection of Fig. 1 shows that approximately twothirds of the CAMP-binding protein, i.e. the regulatory subunit of the protein kinase, was not associated with the CAMPstimulatable protein kinase activity. This fraction of the regulatory subunit was variable, but was usually at least onehalf of the total regulatory subunit. Conversely, there was significant protein kinase activity in the flow-through fraction. This activity reflected the presence of a mixture of CAMP-independent protein kinase(s) and of free catalytic subunit of CAMP-dependent protein kinase. The presence in the mixture of variable amounts of free catalytic subunit was demonstrated by a variable decrease in kinase activity upon the addition of an excess of the mammalian inhibitor (30), specific for CAMP-dependent protein kinase (data not shown) and by detection of the catalytic subunit after passage of the DE52 flow-through fraction through a chromatofocusing column.   Fig. 2 shows the behavior of the pooled fractions with CAMP-dependent protein kinase activity, obtained from the DE52 column, when applied to a Sephadex G-150 column. The protein kinase has an apparent molecular weight of 160,000-180,000 and is stimulated significantly by CAMP. Fig. 3 shows the effect of cAMP concentration on the activity of the D. discoideum holoenzyme. Half-maximal activation is achieved at approximately 30 nM CAMP; the K,,, for ATP is 40 p~ and that for Kemptide 15-25 p~; the optimal M e concentration is 5 mM. The pH range of optimal CAMPdependent protein kinase activity, pH 6.3-8.0, is similar to that found for the mammalian enzyme. GTP at a concentration of 1 mM did not compete with ATP as substrate, irrespective of whether the concentration of ATP was 20 p~ (limiting) or 100 p~; cGMP (100 p~) did not activate the protein kinase.
Dissociation of CAMP-dependent Protein Kinase into Regulatory and Catalytic Subunits-The holoenzyme was dissociated into its subunits by passage through an 8-aminoethylamino-CAMP-Sepharose column as described under "EXperimental Procedures." Regulatory Subunit-The fraction eluted by cAMP contained a major band (280%) at a position corresponding to M, = 41,000 and several minor bands, as visualized by staining with silver. A similar heterogeneity of the CAMP-binding fraction in D. discoideum after one passage through a CAMPaffinity column has been observed by other workers (18,24). The protein with an apparent M, = 41,000 moved to the same position after two-dimensional SDS gel electrophoresis as we reported earlier for the material from crude cytosols or partially purified holoenzyme, photoaffinity-labeled with 8N3-[32P]cAMP (23). After extensive dialysis of the fraction eluted with CAMP, only the protein of M , = 41,000 was photoaffinitylabeled.
Antibody prepared against the regulatory subunit, as described under "Experimental Procedures," was used to react with 8N3-[32P]cAMP photoaffinity-labeled proteins from both crude cytosols and partially purified holoenzyme. In both cases only the M, = 41,000 protein and some fragments, presumably the result of proteolysis during incubation with the antiserum and S. aureus, were precipitated (Fig. 4). Control preparations treated with either preimmune serum obtained from the same rabbit, which was later immunized, or exposed to the photoaffinity label in the absence of light, latory subunit prior to its precipitation. The antibody against the D. discoideum regulatory subunit did not react with bovine Rl, subunit and reacted only poorly with bovine RI subunit (the reactivity was less than 1 / 1~ of that with the D. discoideum subunit). The same lack of cross-reactivity between antisera against the mammalian regulatory subunit and the subunits of Dictyostelium and yeast, respectively, has been observed by other workers (43). The purified regulatory subunit could be phosphorylated in uitro by both the D. discoideum catalytic subunit and purified bovine catalytic subunit. An autoradiogram of a two-dimensional gel of the regulatory subunit phosphorylated by D. discoideum catalytic subunits is shown in Fig. 5. The radioactive spot just on the acid side of the silver-stained regulatory subunit is assumed to be the phosphorylated regulatory subunit. The stained material would then be regulatory subunit, isolated as a less phosphorylated form and not phosphorylated Procedures." The material was solubilized and subjected to onedimensional SDS-gel electrophoresis; the resulting autoradiogram is shown. As controls, the photoaffinity-labeled proteins were submitted to electrophoresis without antibody treatment (Lanes 3 and 4). It is to be noted that the proteins of a molecular weight higher than 41,000 (in Lanes 3 and 4 ) were photoaffinity-labeled nonspecifically; i.e. cAMP at 20 p~ did not compete. These proteins did not react with The area corresponding to the position of the silver-stained regulatory subunit on the same gel has been circled to show its position relative to that of the phosphorylated material. in uitro. No physiological role for this small amount of phosphorylation has been demonstrated and attempts to show phosphorylation of the subunit in uiuo have thus far been unsuccessful.
Catalytic Subunit-The fraction which ran through the CAMP-affinity column and which contained the protein kinase activity was further characterized. Fig. 6A shows the behavior of the protein kinase in a sucrose gradient; a sedimentation value of 2.7 was determined. Sephadex G-150 chromatography of the enzyme (Fig. 6B)  Chromatofocusing of the catalytic subunit (Fig. 7) yielded a PI value of pH 6.4. Fig. 8 indicates that the activity of the catalytic subunit of the CAMP-dependent protein kinase of D. discoideum, but not the activity of CAMP-independent protein kinase, was sensitive to the inhibitor of mammalian CAMP-dependent protein kinase, albeit the concentration of inhibitor required for the inhibition of the slime mold enzyme was significantly higher than that effective for the inhibition of the enzyme of mammalian origin.  and D. discoideum ( -) and of CAMPindependent protein kinase of D. discoideum ( -) . Assay conditions were as described under "Experimental Procedures"; cAMP was employed a t a 1 p~ concentration. 0.1 pl of the preparation contained 100 ng of inhibitor.  7. Chromatofocusing of catalytic subunit. Free catalytic subunit, purified through the CM52 step, was applied to, and eluted from, the column as outlined under "Experimental Procedures." 300pl fractions were collected, and 2 0 4 aliquots were assayed for enzymic activity.

2
FIG. 9. Phosphorylation of bovine regulatory subunits by the D. discoideum catalytic subunit. 3.2 pg of purified bovine RI ( L a n e I ) and 4.2 pg of purified RI1 ( L a n e 2) were phosphorylated in the presence of 5 p~ cAMP by D. discoideum catalytic subunit (purified through the chromatofocusing step) and then submitted to one-dimensional gel electrophoresis; an autoradiogram of the resulting gel is shown.
The substrate specificities of the holoenzyme, purified through the Sephadex G-150 step, and of the free catalytic subunit are compared in Table  I. It may been seen that Kemptide was the most effective substrate for both preparations and the only one where cAMP stimulated significantly the activity of the holoenzyme. Thus, while the specificity of the D. discoideum catalytic subunit for these, nonphysiologi-cal, substrates is not exactly that of the mammalian catalytic subunits, it is clear that the same amino acid sequence (Arg-Arg-X-Ser) is phosphorylated. The selectivity of the D. discoideum catalytic subunit was also tested by its ability to phosphorylate the two bovine regulatory subunits. It may be seen from Fig. 9 that the D. discoideum catalytic subunit, like its mammalian counterpart, catalyzes the phosphorylation of the RII, but not the RI, subunit. This phosphorylation is inhibited by the mammalian inhibitor of CAMP-dependent protein kinase. We have not examined other potential mammalian substrates since we are interested primarily in the isolation of homologous, i.e. D. discoideum, substrates.

DISCUSSION
The findings described here confirm and extend our (15,23) and other investigators' (24,25) earlier observations on the occurrence of a CAMP-dependent protein kinase in the cellular slime mold, D. discoideum. We have isolated and characterized the holoenzyme and its constituent catalytic and regulatory subunits. The enzyme is soluble, at least after disruption of the amoebae by sonic disintegration. The properties of the separated subunits are similar to those which have been described recently by de Gunzburg and Veron (24) and by Rutherford et al. (25). The latter authors also observed that CAMP-stimulatable kinase activity was not adsorbed to DE52 cellulose and had M, = 500,000 whereas we find that the purified holoenzyme has M , = 160,000-180,000. It may be that the preparation employed by Rutherford et al. contained kinase linked to some cytoskeletal component, as has been described for certain preparations of mammalian origin (46, 47). Whereas we did not find a protein kinase of such a high molecular weight, we observed occasionally that a preparation of holoenzyme, activable by CAMP (7-fold activation), was physically undissociated by even millimolar concentrations of CAMP. Photoaffinity-labeling of such preparations with 8Na-[32P]cAMP, followed by SDS-polyacrylamide electrophoresis yielded only the M, = 41,000 regulatory subunit. An investigation of the possibility that the nondissociation of the protein kinase might reflect interaction with a cellular component is currently under way.
In general, the CAMP-dependent protein kinase of D. discoideum resembles the analogous enzyme of vertebrate origin.
The slime mold enzyme has M, = 160,000-180,000, suggesting a tetrameric RzCz structure. Its behavior on the DE52 column ( Fig. 1) is similar to that of the mammalian Type I CAMPdependent protein kinase. Likewise, the kinetic properties of the partially purified CAMP-dependent protein kinase of Dictyostelium are similar to those of the mammalian enzyme, as is its substrate specificity (Table I and Fig. 9); the latter may not be a meaningful criterion in the absence of physiologically relevant substrates.
It appears that the cytosolic D. discoideum CAMP-dependent protein kinase has only one regulatory subunit of M, = 41,000. It seems that the occurrence of only one type of regulatory subunit is the rule in lower eukaryotes, albeit less primitive than D. discoideum; the molecular weight of the regulatory subunit, however, in a number of these fungi is in the range of47,OOO-62,000 (43,48-51). The occurrence of the 41,000 species in the 160,000-180,000 holoenzyme argues that 41,000 is in fact the molecular weight of the native regulatory subunit. Preparation of amoeba1 extracts in the presence of a variety of protease inhibitors and immediate photolabeling of crude cytosols yielded invariably the 41,000 and no higher molecular weight species. Furthermore, purified, photoaffinity-labeled bovine regulatory subunits I and I1 retained their molecular weights (48,000 and 52,000-54,000, respectively) when added to D. discoideum amoebae, and cytosols were then prepared in the usual manner, i.e. by sonic disintegration in the presence of inhibitors of proteolysis. If, however, the extracts were prepared by one cycle of freezing and thawing in the absence of the inhibitors of proteolysis, i.e. under conditions which brought about the degradation of the D. discoideum regulatory subunit to a molecular weight of 32,000-36,000 (63), then the mammalian regulatory subunits were degraded to fragments of molecular weight 37,000-40,000 (data not shown). These findings suggest that proteolysis of the D. discoideum regulatory subunit does not take place under our routine conditions of preparation and there is then no evidence for the occurrence in D. discoideum of regulatory subunits of M, = 48,000-56,000. Until, however, more is known about the precise nature of the subunit interactions that occur in the D. discoideum holoenzyme or until the presence of a blocked NH2 terminus (as occurs in the mammalian proteins) has been demonstrated by amino acid sequencing, the identification of the D. discoideum 41,000 species as the native form of the regulatory subunit must be considered tentative, even though the protein is fully effective in regulating the kinase activity of both the D. discoideum and bovine catalytic subunits. (We made the assumption in the preceding discussion that comparisons of estimates of molecular weights based on SDS-polyacrylamide gel electrophoresis are meaningful in the sense that the molecular weight of the regulatory subunit of the Dictyostelium CAMP-dependent protein kinase is approximately 10,000 lower than that of the analogous bovine subunits. We note, however, that the molecular weights of the bovine regulatory subunits, as derived from amino acid analyses, are much lower than estimates based on the migration of the proteins on SDS gels, i.e. 40,000 for R? and 45,000 for RII (52). Evidently, in those cases SDS does not completely denature the subunits. It is conceivable that the regulatory subunit of the D. discoideum CAMPdependent protein kinase does behave as a globular protein during migration on SDS-polyacrylamide gel electrophoresis and that therefore 41,000 is the actual molecular weight. In that case the difference between the bovine and the slime mold regulatory subunits would be one of conformational form and not of size. Clearly, this question will be answered by a sequencing of the pure Dictyostelium protein kinase regulatory subunit. In the following discussion we shall assume, however, an actual difference in molecular weight).
The properties of the regulatory subunit of the CAMPdependent protein kinase of D. discoideum may be compared with those of the two mammalian subunits. The D. discoideum resembles the RJ subunit in its behavior on DE52 and in the slight cross-reactivity of antibody against the D. discoideum subunit with the bovine RI subunit. There is, however, also a similarity between the D. discoideum and bovine Rrr subunits in that both are eluted from CAMP-affinity columns by 1 mM CAMP; furthermore, the Dictyostelium subunit is a substrate also, albeit a poor one, for autophosphorylation. We note that two-dimensional gel analysis of the regulatory subunit, purified on the CAMP-affinity column, and detected by silverstaining, or of photoaffinity-labeled cytosols (23) invariably reveals at least two spots of M, = 41,000 and with similar PI values. While the two spots may represent modifications of the same gene product, they might also represent the D. discoideum equivalent of Rr and R I I with, in this case, identical molecular weights, but slightly different net charges. The difference in charge would be smaller than that found for the bovine subunits. Here too, comparisons of amino acid sequences should prove instructive. Nonetheless, at the present stage of our knowledge, it appears that the D. discoideum regulatory subunit is not analogous to either mammalian type RI or RII; it may be more meaningful to compare the different regulatory subunits in terms of discrete domains.
Thus, it appears that the CAMP-binding site of the D. discoideum regulatory subunit is similar to, but not identical with, that of the vertebrate CAMP-binding proteins. While there appears to be only 1 CAMP-binding site/molecule of D. discoideum regulatory subunit (24, 53), the relative affinities of several analogs of cAMP for the Dictyostelium regulatory subunit are approximately the same as those for the mammalian regulatory subunits (24, 52). By contrast, the analogs have quite different affinities for the D. discoideum cell surface cAMP receptor (54-56) and cAMP phosphodiesterase (56). By the same token, the affinities of another CAMPbinding protein of M. = 185,000 found in Dictyostelium (14, 18, 57) and a variety of other organisms (58-60) for analogs of cAMP and for derivatives of adenosine are quite different from that of the regulatory subunits of the CAMP-dependent protein kinase. These findings constitute an argument, albeit a tenuous one, for a common evolutionary origin of the D. discoideum and the mammalian CAMP-binding domains as well as conceivably E. coli catabolite gene activator protein (see below). Our earlier findings (15) showed that the regulatory subunit of the D. discoideum CAMP-dependent protein kinase inhibited the activity of the bovine catalytic subunit in a manner reversible by CAMP; this suggests that the domain required for the interaction with the catalytic subunit has been conserved and is presumably similar to that of the homologous mammalian subunit.
The most striking difference between the D. discoideum and the mammalian regulatory subunits is the apparent lack in the D. discoideum regulatory subunit of a significant portion of the polypeptide chain found in the mammalian regulatory subunit. The "extra" M, = 10,000 fragment of the bovine subunits might harbor the second CAMP-binding site which apparently does not occur in the D. discoideum regulatory subunit. Alternatively, the additional amino acid sequences of the mammalian regulatory subunit may play a role in the anchorage of the CAMP-dependent protein kinase to a subcellular structure. While the molecular weight of the D. discoideum catalytic subunit of the CAMP-dependent protein kinase is lower than that of its mammalian analog (61), the affinities of the two catalytic subunits for ATP and for Kemptide, respectively, are similar and the slime mold catalytic subunit catalyzes the phosphorylation of bovine regulatory subunit RII, but not of RI. It is likely, therefore, that the ATP-binding and the protein substrate-binding sites as well as the domains required for interaction with the respective regulatory subunits of the D. discoideum and the mammalian catalytic subunits are similar and derive from the same ancestral domains. There is evidence (13) that D. discoideum represents the deepest known eukaryotic branch of the evolutionary tree and diverged from the path leading ultimately to mammals even before yeasts did; yet, clearly those domains of the CAMPdependent protein kinase with functions, which we were able to test, have been conserved to a high degree. This is not surprising in view of the central role of cAMP in metabolic, hormonal, and developmental regulation. In fact, significant homology in amino acid sequences between the catabolite gene activator protein of the prokaryote E. coli and the RII regulatory subunit of the CAMP-dependent protein kinase of bovine cardiac muscle has been reported recently (62). It appears that the homology resides in the CAMP-binding regions of the two proteins. There is as yet no evidence for the occurrence of amino acid sequences in the eukaryotic regulatory subunits which are homologous t o the DNA-binding domains of the prokaryotic catabolite gene activator protein.
The possibility that such homology exists is probably not ruled out; alternatively, the interaction of the eukaryotic CAMP-binding protein with the catalytic subunit of the protein kinase may fulfill, indirectly, the same physiological function of controlling the synthesis of proteins via the phosphorylation of a relevant protein(s) as does the catabolite gene activator protein by direct interaction with DNA. Clearly the resolution of this question is of crucial importance in arriving at an understanding of the regulation of the synthesis of eukaryotic proteins by CAMP.