Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.     

Neurohormonal receptors and cyclic AMP-binding proteins in rabbit tracheal mucosa-submucosa

Neurohormones and drugs that alter in vitro tracheal electrolyte transport and mucus glycoprotein secretion were examined for their ability to alter cyclic nucleotide accumulation in a smooth muscle-free preparation of rabbit tracheal mucosa-submucosa. cAMP levels were increased by beta-adrenergic agonists, histamine, 2-Cl-adenosine and prostaglandin E1. cGMP levels were increased by carbachol. The phosphodiesterase inhibitor isobutylmethylxanthine increased cAMP and cGMP levels and potentiated only the beta-adrenergic effects. The beta-adrenergic effects were blocked by (+/-)-propranolol and the effects of histamine by diphenhydramine, atropine and (+/-)-propranolol. Atropine blocked the carbachol effects. The isolated surface epithelium from rabbit trachea had higher basal cAMP levels and greater response to beta-adrenergic agonists and isobutylmethylxanthine than the mucosa-submucosa. Two major cAMP-binding proteins in the tracheal mucosa-submucosa were identified with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels also decreased photoaffinity labelling, suggesting that these two cAMP-binding proteins were being occupied in the intact cell. The molecular weights of the proteins were 50 000 and 54 000 and correspond in electrophoretic mobility to the regulatory subunits of Type-I and Type-II cAMP-dependent protein kinases, respectively. The results are consistent with the hypothesis that epithelial functions in the airways are modulated by a number of agonists which increase cyclic nucleotide levels. The effects of beta-adrenergic agonists is apparently mediated by activation of adenylate cyclase and subsequent activation of cAMP-dependent protein kinases.     

Quantitative labeling of the regulatory subunit of type II cAMP-dependent protein kinase from bovine heart by a photoaffinity analog.

Several methods were compared for estimating the amount of regulatory subunit of an 800-fold purified Type II cAMP-dependent protein kinase from bovine heart. These methods included a reversable binding assay using either cAMP, or 8-N3-[32P]cAMP, photoaffinity labeling with 8-N3-[32P]cAMP, and autophosphorylation of the regulatory subunit of the enzyme. Although the regulatory subunit had a slightly lower affinity for 8-N3-cAMP than for cAMP, the total amount of regulatory subunit could be determined by each of the procedures examined. The results indicate that the photoaffinity analog 8-N3-[32P]cAMP is able to label quantitatively all cAMP-binding sites of the regulatory subunit of this cAMP-dependent protein kinase.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Limited proteolysis alters the photoaffinity labeling of adenosine 3',5'-monophosphate dependent protein kinase II with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites. Each regulatory subunit contains two tandem cAMP-binding sites. The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 [Kerlavage, A., & Taylor, S.S. (1980) J. Biol. Chem. 255, 8483-8488]. When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified. One site corresponded to Tyr-381 and, thus, was analogous to the native RII. The other site of modification was identified as Tyr-196, which is not labeled in the native protein. Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites. These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A). These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli.     

Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum

Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.     

Differential androgenic control of prostatic cytosolic cAMP-dependent and -independent protein kinases

Whether or not various cytosolic protein kinases (and especially the type I cAMP-dependent protein kinase) of rat ventral prostate are specifically regulated with respect to total activity or specific activity by androgen has been investigated. Following androgen deprivation, the total activity per prostate of cAMP-dependent protein kinase (with histone as substrate) changed little at 24 h, declining by about 20% at 96 h. Under these conditions, its specific activity remained unaltered at 24 h, but was markedly enhanced at 96 h postorchiectomy. Type II cAMP-dependent protein kinase in rat ventral prostate cytosol was the only form of cAMP-dependent protein kinases present as determined by measurement of catalytic activity as well as [32P]-8-N3-cAMP binding to the regulatory subunits. There was no alteration in the distribution of the isoenzymes of cAMP-dependent protein kinases or the response of these kinase activities to cAMP owing to castration of animals. The prostatic cytosol also contains free regulatory subunit (with molecular weight similar to that of regulatory subunit R1) which coelutes with type II cAMP-dependent protein kinase. This finding was confirmed by using [32P]-8-N3-cAMP photoaffinity labeling of cAMP-binding proteins. With respect to cAMP-independent protein kinase (measured with dephosphophosvitin as substrate), a decline of 31% in its specific activity was observed in cytosol of prostates from rats castrated for a period of 24 h without significant further change at later periods following castration. However, there was a marked progressive reduction in total activity of this enzyme per prostate (loss of 72% at 96 h postorchiectomy). The increase in specific activity of cAMP-dependent, but not cAMP-independent, protein kinase in the face of decreasing total activity in the cytosol at later periods of castration (e.g., at 96 h) may reflect a slower loss of the former enzyme protein than the bulk of the cytosolic proteins. Administration of testosterone to castrated animals prevented these changes. These data do not indicate a specific regulation by steroid of the type I cAMP-dependent protein kinase in the prostate. Rather, the cAMP-independent protein kinase (with dephosphophosvitin as substrate) appears to be modulated by the androgenic status of the animal.     

Multiple cAMP-binding proteins in Aplysia tissues

While it is recognized that cAMP is able to regulate distinct cellular processes differentially, the molecular basis for the diversity of its effects remains unclear. Using photoaffinity labeling with 32P-8 azido-cAMP and two-dimensional gel analysis, we have identified 26 electrophoretic variants of cAMP-binding proteins in the six different tissues of the marine mollusc Aplysia californica sampled. Some of these proteins are found in most tissues, others only in a few; still others appear to be restricted to a single tissue. All of these proteins bind cAMP specifically. The two-dimensional polyacrylamide gel electrophoretic patterns of binding proteins seen in the different tissues fall into three classes. One pattern is shared by the nervous system and embryos. The second is found in muscular tissues (heart, buccal muscle, siphon, and gill). The third pattern is specific to sperm. The presence of distinct subsets of cAMP-binding proteins in different tissues suggests that at least some of the diversity in cAMP's regulatory function may result from diversity in the proteins that bind it.     

Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis.

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.     

Reduced levels of cardiac cAMP-dependent protein kinase in spontaneously hypertensive rat

Cardiac cAMP-dependent protein kinases were compared between the spontaneously hypertensive rat and the age-matched normotensive Wistar-Kyoto rat by DEAE-cellulose chromatography, photoaffinity labeling with 8-N3[32P]cAMP, and Western blots using the antiregulatory and 125I-anticatalytic subunit antibodies. DEAE-cellulose chromatography revealed that the ratio of type I to type II cAMP-dependent protein kinase was 3:1 in the cytoplasmic soluble proteins from the heart of normotensive rat. In contrast, the ratio of type I to type II was 1:1 in the heart of hypertensive rat. Type I protein kinase was reduced by 3-fold in hypertensive rat compared to normotensive rat. The levels of type II protein kinase were similar in both normotensive and hypertensive rats. The ratio of regulatory subunits of type I (RI) to type II (RII) cAMP-dependent protein kinase was 2.5 in the soluble proteins from the heart of normotensive rat compared to a ratio of 0.62 for hypertensive rat. RI was reduced by 4-fold in hypertensive rat compared to normotensive rat. The decrease in RI from hypertensive rat was also demonstrated by photoaffinity labeling with 8-N3[32P] cAMP. Western blot analysis of the catalytic subunit revealed a 2-fold decrease in catalytic subunit (C) in the soluble proteins from the hypertensive rat compared to normotensive rat. These results show that the reduced level of activity of cardiac type I protein kinase in hypertensive rat was the result of a decrease in both the RI and C subunits, thus reducing the number of type I cAMP-dependent protein kinase holoenzyme molecules. Comparison of type I protein kinase from "prehypertensive" and "hypertensive" stages of hypertensive rat indicated that the type I protein kinase was reduced by 3-fold before an increase in the blood pressure was detectable. Cardiac type I protein kinase is predominantly associated with the cytoplasmic proteins in both the normotensive and hypertensive rats. The levels of RI, RII, and C associated with the membrane-solubilized proteins were not affected in the hypertensive rat. The levels of RII were similar in the brain tissue of normotensive and hypertensive rats, suggesting that the decrease in type I protein kinase is specific in hypertensive rat. In conclusion, a decrease in cardiac type I cAMP-dependent protein kinase may affect the degree of phosphorylation of cardiac regulatory proteins, thus impairing normal cardiac physiology in hypertensive rat.     

Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.     

Induction of cAMP-dependent protein kinase I during human monocyte differentiation

Differentiation of human peripheral blood monocytes into macrophages was accompanied by induction of the regulatory subunit of cAMP-dependent protein kinase I as determined by photoaffinity labeling of cytosol proteins with 8-N3-[32P]cAMP and DEAE-Sephacel chromatography. The appearance of cAMP-dependent protein kinase I in macrophages was not due to translocation from the particulate fraction of monocytes. The regulatory subunit of cAMP-dependent protein kinase II was present in both monocytes and in vitro-differentiated macrophages. Protein kinase I in macrophages demonstrated higher affinity for 8-N3-cAMP (KD = 0.7 nM) than did protein kinase II from either monocytes (KD = 14.5 nM) or macrophages (KD = 4.9 nM). These studies demonstrate induction of the regulatory subunit of cAMP-dependent protein kinase I during the differentiation of a normal human cell and support the hypothesis that cAMP may regulate some stages of differentiation.     

Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae

The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control. The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting. The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction. By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.     

Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.     

Distinctive patterns of cAMP-binding proteins and their relationship to hemopoietic cellular differentiation

In order to clarify distinctions among cAMP-binding protein patterns present in hemopoietic cells of the various major lineages, we have studied extracts from 18 human cell lines by photoaffinity labelling with 8-azido [32P]cAMP, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Four major cAMP-binding protein bands were noted. These occurred in five recognizable patterns of combinations, each of which was restricted to cells of particular lineages. A distinctive pattern was found in the pluripotent stem cell line K562, confirming its unusual nature compared with other lines committed to myeloid or lymphoid differentiation. Three patterns were noted among the pre-B and early and late B cell lines studied, which may thus define sequential stages of differentiation of this series. These studies indicate the utility of cAMP-binding proteins as a biochemical differentiation marker system. The variety of phenotypes noted further suggests a role for them in the development or expression of specialized cellular functions.     

A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.