CAMP mediated cell growth regulation. I. Characterization of protein kinases in CHO cells

Protein kinases in wild-type CHO cells have been characterized. Cells cultured on MEM were collected, homogenized and the extract assayed for protein kinase activity. DEAE cellulose chromatography of 30.000xg extract yields 2 peaks of protein kinases activity, PKI and PKII. The two peaks when analyzed for the binding of 8-N3-(32P)cAMP show two subunits RI and RII and a RI not associated with the enzymatic activity, named RF. This characterization allows us to discuss the meaning of protein kinases in the modulation of the growth regulating effects of cAMP.     

A study of cAMP binding proteins on intact and disrupted sperm cells using 8-azidoadenosine 3',5'-cyclic monophosphate

The photoaffinity probe (32P) 8-N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP-dependent protein kinase (cAMP-PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell-free seminal plasma was found to be free of detectable (32P) 8-N3 cAMP-binding proteins. The 8-N, cAMP was also effective in stimulating endogenous cAMP-PK activity in intact and disrupted sperm. A substantial amount of (32P) 8-N3 cAMP binding to types I and II regulatory subunits and cAMP-PK activity was detected on washed intact cells. Intact cells bound 1.80 pmol of (32P) 8-N3 cAMP/mg protein and had cAMP-PK activity of 824 units/10(8) cells. Disrupted cells bound 3.95 pmol (32P) 8-N3 cAMP/mg protein and had a cAMP-PK activity of 2,206 units/10(8) cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8-N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.     

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.     

Protein kinases in developing rat brain.

The levels of cAMP-dependent protein kinases were measured in developing rat brain by a variety of methods. The regulatory subunit (R) was measured both by [3H]cAMP binding and by 8-N3-[32P]cAMP incorporation. The catalytic subunit (C) was measured by an assay of histone kinase activity. Data were calculated per mg protein. Neither R nor C levels changed significantly in either membranes or cytosol during development. The ratio of R to C was essentially unity in the cerebra of both newborn (2-day-old) and adult (40-day-old) rats. Polyacrylamide-gel electrophoresis resolved two regulatory subunits (R-I) and (R-II) which were derived from the Type I and Type II cAMP-dependent protein kinases, respectively. 8-N3-[32P]cAMP incorporation into Proteins R-I and R-II indicated that the amounts of Proteins R-I and R-II did not change significantly in either membranes or cytosol during development.     

Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the cAMP receptor protein (CRP) of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has been demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analogue. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5'AMP. The data indicate that (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, one-third remains associated with the amino-proximal alpha core region of CRP [Eilen, E., Pampeno, C., & Krakow, J.S. (1978) Biochemistry 17, 2469] which contains the cAMP binding domain; the remaining two-thirds of the label associated with the beta region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the molecular weight 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. These results suggest that a part of the carboxyl-proximal region is folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.     

Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabeling of holoenzyme I with 8-azidoadenosine 3',5'-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N6-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-371 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of nucleotide photoaffinity probes to study hormone action

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.     

Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.

We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.     

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.     

Photoaffinity labeling of adenosine 3',5'-cyclic monophosphate binding sites of human red cell membranes.

To identify and investigate the cAMP binding sites of human red cell membranes a photoaffinity analog of cAMP, 8-azidoadenosine 3',5'-cyclic monophosphate (8-N3cAMP), has been synthesized. This analog activates cAMP-dependent protein kinase(s) in the red cell membrane. It exhibits tight, but reversible binding to the membranes which is competitive with cAMP. Photolysis of [32P]-8-N3cAMP with red cell membranes results in covalent incorporation of radioactive label onto two specific membrane proteins. This incorporation requires activating light and is reduced to background levels with addition of low levels of cAMP. Prephotolysis of 8-N3cAMP completely abolished its ability to photolabel membrane proteins. Both the reversible and photocatalyzed binding of 8-N3cAMP show saturation kinetics. The molecular weights of the two primarily labeled proteins are approximately 49,000 and 55,000. The differential effects of cAMP, ATP, and adenosine on the photocatalyzed incorporation of [32P]-8-N3cAMP onto these two proteins suggest that they have biochemically different properties. The potential usefulness of this compound for investigating various molecular aspects of cAMP action is discussed.     

Post-translational modifications of the regulatory subunits of cAMP-dependent protein kinases during G1/S progression

During the G1/S transition of the cell cycle variations in the labelling by 8-N3-[32P]cAMP of the protein kinase A regulatory subunits RI and RII, used as a probe to monitor post-translational modifications that may regulate cAMP binding, were observed in synchronized HeLa cells. A decrease in 8-N3-[32P]cAMP labelling of RI, RII and RII phosphorylated by the catalytic subunit of PKA was correlated with the increased percentage of cells in phases G1. An increase in 8-N3-[32P]cAMP incorporated into the 54-kDa RII subunit during progression from G1 to S was correlated with an increase in intracellular cAMP. A transient increase in Mn-SOD activity was detected in cells arrested at the G1/S transition using two different techniques, suggesting that oxidative modulation of regulatory subunits by free radicals may modify cAMP binding sites during the cell cycle. Decreased photoaffinity labelling by 8-N3-[32P]cAMP of RI, RII and autophosphorylated RII subunits was found to be an inherent characteristic of PKA in the G1/S transition.     

DNA-mediated gene transfer of a mutant regulatory subunit of cAMP-dependent protein kinase

We have used DNA-mediated gene transfer of genomic DNA to introduce into wild-type Chinese hamster ovary (CHO) cells a mutant gene that confers resistance to the growth inhibitory effect of cAMP. This dominant mutation in CHO cell line 10248 is responsible for an alteration in the RI subunit (RI*) of the type I cAMP-dependent protein kinase (Singh, T. J., Hochman, J., Verna, R., Chapman, M., Abraham, I., Pastan, I.H., and Gottesman, M.M. (1985) J. Biol. Chem. 260, 13927-13933). The transformant 11564 which was studied in detail, has the same characteristics as the original mutant 10248 including continued growth in medium containing 8-Br-cAMP, an increase in the Ka for cAMP activation of the kinase, a greatly reduced amount of type II protein kinase activity, an altered incorporation of the photoaffinity label 8-N3[32P]cAMP into the RI* subunit of PKI, and an absence of cAMP-dependent phosphorylation of a Mr = 52,000 protein in intact cells. In addition, analysis of the DNA of the transformant indicates the presence of an increased amount of DNA for the RI gene. These results are consistent with the transfer of a mutant gene for the RI* subunit of the cAMP-dependent protein kinase and its phenotypic expression in the transformant and also support the hypothesis that the mutation responsible for the defect in cell line 10248 is due to an alteration in the gene for RI.     

Photoaffinity labeling of a protein kinase from bovine brain with 8-azidoadenosine 3',5'-monophosphate.

8-Azidoadenosine 3',5'-monophosphate (8-N3-cAMP) containing 32P has been used as a photoaffinity label specific for the adenosine 3',5'-monophosphate (cAMP) binding site(s) present in a partially purified preparation of soluble protein kinase from bovine brain. 8-N3-cAMP and cAMP were found to compete for the same binding site(s) in this preparation, as determined by a standard filter assay. When this protein preparation was equilibrated with [32P]-8-N3-cAMP, and then irradiated at 253.7 nm, the incorporation of radioactivity was predominantly into a protein with an apparent molecular weight of 49,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. This labeled protein comigrated in the gel with the only protein which is endogenously phosphorylated by [gamma-32P]ATP, a protein which has been shown to be the regulatory subunit of the protein kinase (H. Maeno, P. L. Reyes, T. Ueda, S. A. Rudolph, and P. Greengard (1974), Arch. Biochem. Biophys. 164, 551). The incorporation of [32P]-8-N3-cAMP into this protein was half-maximal at a concentration of 7 x 10(-8) M. In accordance with a proposed mechanism involving the formation of a highly reactive nitrene intermediate upon irradiation of the azide, the incorporation of radioactivity into protein was maximal within 10 min of irradiation, and was almost eliminated by preirradiation of the photolabile ligand. Moreover, this incorporation was virtually abolished by a 50-fold excess of cAMP, but not by AMP, ADP, ATP, or adenosine. We suggest that 8-N3-cAMP may prove to be a useful molecular probe of the cAMP-binding site in receptor proteins and report its use in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a highly sensitive and selective radiochemical marker for cAMP-binding proteins.     

Photoaffinity labeling of the cell surface adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum and its modification in down-regulated cells

The cAMP cell surface receptor of Dictyostelium discoideum amoebae was identified by the use of the photoaffinity analogue 8-N3-[32P]cAMP. Labeling by intact cells of one component, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, could be specifically inhibited by the presence of nonradioactive cAMP. The component, P45 (apparent molecular weight of 45,000), was not identified on vegetative cells but was labeled with increasing intensity as cells differentiated and increased their levels of surface cAMP binding sites. Developmental mutants, starved under conditions where they do not express significant levels of cAMP binding sites, did not incorporate radioactivity into this protein. These mutants did label P45 when starved under differentiation-inducing conditions such that their levels of surface cAMP binding sites increased. P45 co-purified with the plasma membrane fraction isolated from cells to which 8-N3-[32p]cAMP had been covalently bound. Down-regulated amoebae, which displayed approximately 25% of the binding activity of untreated cells, did not label P45. These cells did, however, label a new component with an apparent molecular weight of 47,000 (P47).l The appearance of this component represented the only discernible difference in labeling profile under these conditions. As in the case of P45, radioactive incorporation into P47 did not occur if the photoactivation of 8-N3-[32P]cAMP was performed in the presence of nonradioactive cAMP.     

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.     

Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate

Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine 3',5'-monophosphate-receptor protein and protein kinase in Coprinus macrorhizus

A single cAMP-receptor protein could be detected in mycelial extracts of Coprinun macrorhizus by using the photoaffinity cAMP-analogue, 8-N3-cAMP. The protein which specifically bound 32P-labeled 8-N3-cAMP had an apparent molecular weight of 46,000 as determined by an SDS-polyacrylamide gel electrophoresis system. The 46,000-dalton protein was characterized by the dissociation constant for [32P]-8-N3-cAMP, and by the nucleotide specific inhibition of [32P]-8-N3-cAMP binding. The 46,000-dalton protein was co-chromatographed on a DEAE-cellulose column with cAMP-dependent protein kinase. The levels of [32P]-8-N3-cAMP-binding and protein kinase activities in mycelial extracts of strains used was always in parallel. The result indicated that the 46,000-dalton protein may be a regulatory subunit of protein kinase with the capacity to bind cAMP. cAMP-dependent protein kinase of this fungus was immunologically different from those of higher animals.     

A nuclear cAMP binding protein in retinoic acid-treated HL-60 cells

A cAMP binding protein was detected in HL-60 cells using photoaffinity labeling with 8-azido [32P]cAMP. The binding protein was found in a 0.35 M NaCl nuclear protein extract from untreated HL-60 cells and from the HL-60 cells induced to mature with retinoic acid. While the quantity of the cAMP binding protein did not change following the induced differentiation, a second form of the subunit, altered in charge, was present at 3 and 5 days after retinoic acid treatment. The findings indicate that the regulatory subunit of the type II cAMP-dependent protein kinase could be involved in nuclear functions associated with human myeloid cell differentiation.     

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.     

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.