Occurrence of a cyclic AMP-dependent protein kinase on the outer surface of rat epididymal spermatozoa.

Endogenous protein kinase activity was detected on the outer surface of rat cauda epididymal spermatozoa. The kinase activity of the intact sperm cells catalyses the transfer of the terminal phosphate of exogenous [γ³²-P] ATP to the alkali labile phosphoester bonds of exogenous calf thymus histones. There was little uptake of [γ³²-P] ATP and phosphorylation of endogenous proteins by intact spermatozoa. The amount of histones phosphorylated by the peripherial kinase is directly proportional to the sperm numbers and the reaction is linear for approx. 5 min. Cyclic AMP (2.5 μM) activates the kinase (approx. 120%) and also causes the release of the enzyme from spermatozoa into the medium. Approx. 80% of the peripherial kinase activity is released after 30 seconds of incubation of spermatozoa.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Cyclic AMP-dependent protein kinase from Ustilago maydis

Summary Ustilago maydis was surveyed for cyclic AMP-dependent protein kinase activity. Using a combination of ion-exchange and molecular filtration techniques, we demonstrate that there is only one form of cyclic AMP-dependent protein kinase in the cytosolic fraction of the fungus. The kinase activity is specifically activated by cyclic AMP and utilizes protamine and kemptide as substrates. Most, if not all, of the cyclic AMP binding detected in the soluble fraction is associated with the protein kinase activity. Cyclic AMP-dependent protein kinase is completely dissociated by cyclic AMP into catalytic and regulatory subunits having an apparent molecular weight of 35 000 daltons as judged by sucrose gradient centrifugation.Post graduate fellow from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).Career investigator from CONICET.     

Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function

Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP-dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance.     

Movement characteristics of bovine epididymal spermatozoa: effects of forward motility protein and epididymal maturation

Movement characteristics of untreated bovine caudal epididymal spermatozoa were compared by high-speed cinemicrography with those of theophylline-activated caput epididymal spermatozoa with and without added forward motility protein (FMP). Comparison of individual movement characteristics clearly established the importance of FMP in converting the nonprogressive motility of theophylline-activated caput sperm into the progressive swimming of mature caudal sperm. Although the total or curvilinear distance traveled in 1 sec by theophylline-activated caput sperm was not changed by the addition of FMP, the linear progression was doubled and the percentage of progressively motile sperm was tripled by this protein. Untreated caudal sperm were 80% motile and theophylline-activated caput sperm were nearly 50% motile; the percentage of motile sperm that were progressive was the same for theophylline-activated caput sperm with FMP and for untreated caudal sperm. Caput sperm without FMP roll infrequently, if at all, but caput sperm with FMP and caudal sperm roll at 4.7 Hz. The beat frequency increases significantly with the addition of FMP and is even higher for caudal sperm. The hydrodynamic power output rises concomitantly with the beat frequency. Perhaps the most striking difference between caput sperm without FMP and those with it is in the swimming paths they follow. Caput sperm without FMP exhibit frequent reversals in direction, or yawing of the sperm heads as they loop back and cross over their tails in an apparently very flexible bending. Their average swimming paths are circles. Caput sperm with FMP and caudal sperm do not show this behavior, but swim in average paths which are linear. The minimum radius of curvature of the tail of caput sperm without FMP is much smaller than that for the other two cell types. These studies clarify the role of FMP in epididymal development of sperm motility.     

Cyclic nucleotide selectivity of protein kinase G isozymes

The intrinsic activity of the C‐terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)‐dependent protein kinases (PKG) is inhibited by interactions with the N‐terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R‐domain disrupts the inhibitory R–C interaction, leading to the release and activation of the C‐domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C‐domain are more cGMP selective and therefore critical for cGMP‐dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme‐specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB‐mediated dimeric contacts that indicate cGMP‐driven reorganization of domain–domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.     

Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.     

Cyclic AMP-dependent protein kinase regulates the subcellular localization of Snf1-Sip1 protein kinase

The Snf1/AMP-activated protein kinase family has diverse roles in cellular responses to metabolic stress. In Saccharomyces cerevisiae, Snf1 protein kinase has three isoforms of the beta subunit that confer versatility on the kinase and that exhibit distinct patterns of subcellular localization. The Sip1 beta subunit resides in the cytosol in glucose-grown cells and relocalizes to the vacuolar membrane in response to carbon stress. We show that translation of Sip1 initiates at the second ATG of the open reading frame, yielding a potential site for N myristoylation, and that mutation of the critical glycine abolishes relocalization. We further show that the cyclic AMP-dependent protein kinase (protein kinase A [PKA]) pathway maintains the cytoplasmic localization of Sip1 in glucose-grown cells. The Snf1 catalytic subunit also exhibits aberrant localization to the vacuolar membrane in PKA-deficient cells, indicating that PKA regulates the localization of Snf1-Sip1 protein kinase. These findings establish a novel mechanism of regulation of Snf1 protein kinase by the PKA pathway.     

Cyclic AMP-dependent protein kinase does not phosphorylate cyclic GMP-dependent protein kinase in vitro

The autophosphorylation reaction of purified cGMP-dependent protein kinase has been studied. Apparent initial rates of autophosphorylation in the absence of cyclic nucleotides and in the presence of cGMP and cAMP are 0.006, 0.04, 0.4 mol Pi incorp./min-1. mol cGMP-kinase subunit-1. In the presence of cGMP and cAMP approximately 1 and 2 mol Pi are incorporated/mol enzyme subunit. These values are independent of the enzyme concentration. Stimulation of autophosphorylation by cAMP is not due to activation of a contaminating cAMP-dependent protein kinase since: (a) addition of the heatstable inhibitor protein of cAMP-kinase does not inhibit autophosphorylation; and (b) catalytic subunit of cAMP-kinase added at a 10-fold excess over cGMP-kinase does not phosphorylate cGMP-kinase.     

Cyclic AMP-dependent protein kinase in canine pancreatic rough endoplasmic reticulum

A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.     

Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin.

We previously observed that Ser378 in the heparin-binding domain of vitronectin becomes phosphorylated by a protein kinase in plasma upon addition of ATP and divalent cations. We now report that purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein and that vitronectin becomes phosphorylated during biosynthesis in human hepatoma (HepG2) cells. In vitro, rabbit muscle cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain (75 kDa) vitronectin but does not phosphorylate the two-chain (65/10 kDa) form cleaved at Arg379. Heparin affects neither the time course nor the extent of phosphorylation of Ser378 at neutral pH. The extent of phosphorylation of Ser378 achieved with cAMP-dependent protein kinase (greater than or equal to 0.3 mol phosphate per mol vitronectin) is greater than that obtainable in plasma and should enable comparisons to be made of the activities of the native and phosphorylated forms.     

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.     

Evidence against phosphorylation of a cyclic AMP-dependent protein kinase from bovine tracheobronchial smooth muscle.

The data presented in this report are evidence against the autophosphorylation of the cyclic AMP-dependent protein kinase isolated from bovine tracheobronchial smooth muscle. This suggests that there may be a fundamental difference in the regulation in vivo of the protein kinases from bovine heart and tracheobronchial smooth muscle.     

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.     

Cyclic AMP-dependent protein kinase activity and protein phosphorylation in zones of the adrenal cortex

Steroidogenesis is not stimulated by ACTH in the inner zone of the guinea pig adrenal cortex; adenylate cyclase is normally stimulated. To further explore the lack of a steroidogenic response to ACTH in the inner zone, cAMP-dependent protein kinase activity and protein phosphorylation were examined in the outer and inner adrenocortical zones. To summarize: total cAMP-dependent protein kinase activity was 40% higher in the outer zone than in the inner zone; of the total cAMP-dependent protein kinase activity, cytosol contained 80% for the outer and 70% for the inner zone. In both zones only the type II isozyme was present. Qualitative and quantitative differences in protein phosphorylation were noted for the two zones.