cAMP-independent Protein Kinase Research Articles

PD03-02 NOVEL STRUCTURAL VARIANTS IN CTNNB1 AND MOLECULAR CLASSIFICATION IN CORTISOL-PRODUCING ADENOMA

You have accessJournal of UrologyCME1 Apr 2023PD03-02 NOVEL STRUCTURAL VARIANTS IN CTNNB1 AND MOLECULAR CLASSIFICATION IN CORTISOL-PRODUCING ADENOMA Yoichi Fujii, Seiichiro Higuchi, Yusuke Sato, Yuichi Shiraishi, Kenichi Yoshida, Satoru Miyano, Haruki Kume, Tomohiko Ichikawa, Atsushi Iwama, Tomoaki Tanaka, and Seishi Ogawa Yoichi FujiiYoichi Fujii More articles by this author , Seiichiro HiguchiSeiichiro Higuchi More articles by this author , Yusuke SatoYusuke Sato More articles by this author , Yuichi ShiraishiYuichi Shiraishi More articles by this author , Kenichi YoshidaKenichi Yoshida More articles by this author , Satoru MiyanoSatoru Miyano More articles by this author , Haruki KumeHaruki Kume More articles by this author , Tomohiko IchikawaTomohiko Ichikawa More articles by this author , Atsushi IwamaAtsushi Iwama More articles by this author , Tomoaki TanakaTomoaki Tanaka More articles by this author , and Seishi OgawaSeishi Ogawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003220.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cortisol-producing adenoma (CPA) is the most common hormone-producing adrenal tumor. Although several hotspot mutations of driver genes (PRKACA, GNAS, and CTNNB1) have been identified in the previous studies, genotypes have not been confirmed in approximately 30% of CPAs. In addition, the association between genotypes and clinical characteristics of CPAs are poorly understood. METHODS: Genomic DNA and RNA was extracted from 125 surgical specimens of adrenal CPAs, including 60 cases with Cushing’s syndrome (CS) and 65 with subclinical Cushing’s syndrome (SCS), and subjected to targeted-capture sequencing and RNA sequencing. RESULTS: Novel structural variants (SVs) in CTNNB1, including long deletion and inversion, were identified in 8% (10/125) of CPA cases. All these SVs resulted in the lack of exon 3, which encodes the phosphorylation site of beta-catenin and plays an important role in its degradation, leading to the constitutive activation of the Wnt pathway by disrupting the degradation of beta-catenin. In addition, several mutations in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA), were identified around the known hotspot p.L206. Subsequent functional assay showed that these PRAKACA mutants abolished its binding ability to PRKAR1A, the regulatory subunit of PKA, leading to the constitutive, cAMP-independent PKA activation. In total, 82.4% (103/125) of CPAs had alterations in PRKACA, GNAS, and wnt pathway genes (CTNNB1, APC, and MED12) in almost mutually exclusive manner, based on which CPAs are classified into three distinct subgroups. These mutational subtypes showed distinct genetic and clinical characteristics. RNA sequencing revealed that the expression level of steroidogenic enzymes such as CYP17A1 and CYP21A2 were more upregulated in PRKACA-mutated subtype than wnt-mutated subtype. Consistent with this observation, PRKACA-mutated subtype showed significantly higher cortisol-producing autonomous secretion level, more frequent CS than wnt-mutated subtype. In contrast, wnt-mutated subtype more tended to be SCS, showed lower cortisol-producing ability and largar tumor volume compared to the other two subtypes. CONCLUSIONS: Novel CTNNB1 structural variants were identified. CPAs showed distinct genetic landscape, associated with various clinical features. Source of Funding: No © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e74 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoichi Fujii More articles by this author Seiichiro Higuchi More articles by this author Yusuke Sato More articles by this author Yuichi Shiraishi More articles by this author Kenichi Yoshida More articles by this author Satoru Miyano More articles by this author Haruki Kume More articles by this author Tomohiko Ichikawa More articles by this author Atsushi Iwama More articles by this author Tomoaki Tanaka More articles by this author Seishi Ogawa More articles by this author Expand All Advertisement PDF downloadLoading ...

Glucose deprivation activates a cAMP-independent protein kinase from Trypanosoma equiperdum.

Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07-51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22-1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm, respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, β,γ-imidoadenosine 5'-triphosphate and 5'-[p-(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI-α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.

PD3-06 IMPACT OF PRIMARY HISTOLOGY ON DISEASE FREE SURVIVAL AFTER MINIMALLY INVASIVE ADRENALECTOMY FOR METASTATIC CANCER

INTRODUCTION AND OBJECTIVES: Cushing’s syndrome is characterized by excess cortisol production from adrenocortical grand and presents various symptoms and signs. In corticotropin-independent Cushing’s syndrome, the excess cortisol production is attributed to an adrenocortical adenoma in the majority of cases, in which the underlying molecular pathogenesis has been poorly understood. METHODS: Somatic mutations in 8 adrenocortical tumors associated with corticotropin-independent Cushing’s syndrome was explored by whole exome sequencing, followed by target Sanger sequencing in an additional 57 cases. Functional impacts of detected mutations were also investigated by in vitro binding assays and assays for kinase activity. RESULTS: We identified novel hotspot mutation in PRKACA which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). PRKACA mutations were found in 34 (52.3%) of 65 patients and invariably resulted in L206R variant protein. We also detected activating mutations of G-protein subunit as (GNAS) (R201C and R201H) in an additional 11 (16.9%) patients, which lead to constitutive accumulation of cAMP. Mutations in PRKACA and GNAS were completely mutually exclusive and accounted for 70% of samples had mutations in genes involved in the cAMP/PKA signaling pathway. The L206R PRKACA mutant abolished its binding to the regulatory subunit of PKA which inhibits catalytic activity of PKA and lead to constitutive, cAMP independent PKA activation. CONCLUSIONS: Corticotropin-independent Cushing’s syndrome is frequently associated with somatic mutations in PRKACA or GNAS. The current results highlight the major role of constitutive activation of the cAMP/PKA signaling pathway by somatic mutations in Corticotropin-independent Cushing’s syndrome and provide novel insight into the diagnosis and treatment of this unique syndrome.

Recurrent somatic mutations underlie corticotropin-independent Cushing's syndrome.

Cushing's syndrome is caused by excess cortisol production from the adrenocortical gland. In corticotropin-independent Cushing's syndrome, the excess cortisol production is primarily attributed to an adrenocortical adenoma, in which the underlying molecular pathogenesis has been poorly understood. We report a hotspot mutation (L206R) in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), in more than 50% of cases with adrenocortical adenomas associated with corticotropin-independent Cushing's syndrome. The L206R PRKACA mutant abolished its binding to the regulatory subunit of PKA (PRKAR1A) that inhibits catalytic activity of PRKACA, leading to constitutive, cAMP-independent PKA activation. These results highlight the major role of cAMP-independent activation of cAMP/PKA signaling by somatic mutations in corticotropin-independent Cushing's syndrome, providing insights into the diagnosis and therapeutics of this syndrome.

5-Hydroxyeicosatetraenoic acid and leukotriene D4 increase intestinal epithelial paracellular permeability

The loss of epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease, with levels of 5-lipoxygenase metabolites being increased in the mucosa of these patients. The objective of this study was to determine the effect of the eicosanoids produced by the 5-lipoxygenase pathway, leukotriene B4 and D4, and 5-hydroxyeicosatetraenoic acid on epithelial barrier function. Paracellular permeability was estimated from fluorescein isothiocyanate–dextran fluxes and transepithelial electrical resistance in differentiated Caco-2 cells. Our results suggest that leukotriene D4 and 5-hydroxyeicosatetraenoic acid altered both parameters. Identification of the receptors involved in these changes indicated that cysteinyl-leukotriene receptor 1 participates in the effects of leukotriene D4. For both eicosanoids, these effects were mediated by activation of the phospholipase C/Ca2+/protein kinase C pathway, in addition to cAMP-independent protein kinase A activation. Furthermore, we observed a correlation between increased paracellular permeability and the redistribution of occludin, and for leukotriene D4, the disorganization of the subapical actin ring and myosin light chain kinase activation. In conclusion, on the basis of our results, we propose that 5-lipoxygenase pathway metabolites participate in the disruption of epithelial barrier function that is characteristic of inflammatory bowel disease.

Analyses of the Involvement of PKA Regulation Mechanism in Meiotic Incompetence of Porcine Growing Oocytes1

Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms.

Functional regulation of L-type calcium channel via a novel cAMP-independent protein kinase (CIK) in goat epididymal spermatozoa

Functional regulation of L-type calcium channel via a novel cAMP-independent protein kinase (CIK) in goat epididymal spermatozoa

Gene Deletion of 7,8-Linoleate Diol Synthase of the Rice Blast Fungus: STUDIES ON PATHOGENICITY, STEREOCHEMISTRY, AND OXYGENATION MECHANISMS

Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Delta7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Delta7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Delta mac1 sum1-99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.

B Subunits of Shiga Toxin 2 Induce VWF Secretion by a cAMP-Independent Protein Kinase A Signaling Pathway.

Abstract 3046Poster Board II-1022Shiga toxin (Stx) is composed of an A subunit that inactivates ribosomes and 5 B subunits that bind globotriaosylceramice (Gb3) on cell surfaces to facilitate internalization and retrograde transport of the toxin to the cytosol. Stx-producing E. coli cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. In animal experiments, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice by inducing the secretion of von Willebrand factor (VWF). Using cultured human umbilical vein endothelial cells (HUVECs), we have shown that the B subunits of either Stx1 or Stx2 are sufficient to stimulate VWF secretion, independent of the catalytically-active A subunit. HUVECs were perfused in a parallel plate flow chamber with Stx preparations and fluorescently-labeled anti-VWF, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Either Stx1B or Stx2B rapidly induced the secretion of long strings of VWF that attached to the cell surface and bound platelets with high affinity. VWF string formation was maximal within 5 min and was blocked by soluble analogs of Gb3 or anti-Stx B subunit antibodies. Stx1B subunits activated phospholipase C (PLC), increased intracellular Ca2+, and activated protein kinase C (PKC). Stx2B is homologous to Stx1B, but their amino acid sequences differ by 34% and Stx2B activates distinct signaling pathways. For example, pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), Ca2+-free medium (pH 7.2 PBS, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min) or a protein kinase C inhibitor (5 μM 19-31, 1 h) blocked VWF secretion in response to Stx1B, but not Stx2B. The role of intracellular Ca2+ was also assessed using a fluorescent BD Calcium Assay. Stx1B (5 nM), histamine (0.1 mM) or thrombin (1 U/ml) induced a similar transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min, whereas Stx2B did not affect the intracellular Ca2+ level. Conversely, pretreatment with a protein kinase A inhibitor (5 μM H89, 30 min) had no effect on Stx1B-induced VWF string formation, but decreased Stx2B-induced VWF string formation more than 90%. Neither Stx1B nor Stx2B increased intracellular cAMP in cell extracts within 5 min. To more directly detect cAMP production, HUVECs were transfected with a CFP-Epac-YFP construct that exhibits decreased FRET upon binding cAMP, which is detected by confocal microscopy. Forskolin caused the expected increase in CFP/YPF ratio, indicating a rise in cAMP, whereas Stx1B and Stx2B did not. Despite the absence of a change in cAMP, Stx2B caused an increase in PKA activity comparable to that induced by forskolin, when assayed in cell extracts with a synthetic peptide substrate (Assay Designs) kit. In addition, treatment of HUVECs with Stx2B increased the phosphorylation of the endogenous PKA substrate, 14-3-3ζ. These results indicate that Stx2B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3 and cAMP-independent activation of PKA. Therefore, Stx2 may contribute to the pathogenesis of HUS by at least two mechanisms that affect microvascular endothelium: Stx2A subunit-dependent inhibition of protein synthesis, and Stx2B subunit-dependent induction of VWF secretion. DisclosuresSadler:Baxter BioSciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ablynx: Consultancy.

Dynamic Binding of PKA Regulatory Subunit RIα

Recent crystal structures have revealed that regulatory subunit RIalpha of PKA undergoes a dramatic conformational change upon complex formation with the catalytic subunit. Molecular dynamics studies were initiated to elucidate the contributions of intrinsic conformational flexibility and interactions with the catalytic subunit in formation and stabilization of the complex. Simulations of a single RIalpha nucleotide binding domain (NBD), missing cAMP, showed that its C helix spontaneously occupies two distinct conformations: either packed against the nucleotide binding domain as in its cAMP bound structure, or extended into an intermediate form resembling that of the holoenzyme structure. C helix extension was not seen in a simulation of either RIalpha NBD. In a model complex containing both NBDs and the catalytic subunit, well-conserved residues at the interface between the NBDs in the cAMP bound form were found to stabilize the complex through contacts with the catalytic subunit. The model structure is consistent with available experimental data.

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases.

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.

Protein kinases and their protein substrates in free messenger ribonucleoprotein particles and polysomes from mouse plasmacytoma cells.

In free messenger ribonucleoprotein particles (mRNP) and polysomes from plasmacytoma cells, a phosphorylated protein/protein kinase system has been characterized by a combination of oligo(dT)-cellulose chromatography and CsCl isopycnic gradient centrifugation. The presence phosphorylated in vivo has been detected in both types of particles. Endogenous protein phosphorylation occurs in vitro by particle-associated cAMP-independent protein kinase(s) using [gamma-32P]ATP and [gamma-32P]GTP. These kinases are sensitive to hemin action. Analysis of mRNP proteins by gel electrophoresis and autoradiography showed strong analogies between the phosphorylation patterns obtained in vivo and in vitro, suggesting a substrate specificity for the associated enzymes. The phosphorylated proteins have been compared to initiation factors and ribosomal proteins. We have partially purified the cAMP-independent protein kinase activities responsible for the endogenous phosphorylation in free mRNP and polysomes; two activities were identified in free mRNP whereas three activities were found to be associated with polysomes.

Adenosine A 2A receptor facilitation of hippocampal synaptic transmission is dependent on tonic A 1 receptor inhibition

Adenosine tonically inhibits synaptic transmission through actions at A 1 receptors. It also facilitates synaptic transmission, but it is unclear if this facilitation results from pre- and/or postsynaptic A 2A receptor activation or from indirect control of inhibitory GABAergic transmission. The A 2A receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission in the CA1 area of rat hippocampal slices (by 14%), independent of whether or not GABAergic transmission was blocked by the GABA A and GABA B receptor antagonists, picrotoxin (50 μM) and CGP 55845 (1 μM), respectively. CGS 21680 (10 nM) also inhibited paired-pulse facilitation by 12%, an effect prevented by the A 2A receptor antagonist, ZM 241385 (20 nM). These effects of CGS 21680 (10 nM) were occluded by adenosine deaminase (2 U/ml) and were made to reappear upon direct activation of A 1 receptors with N 6-cyclopentyladenosine (CPA, 6 nM). CGS 21680 (10 nM) only facilitated (by 17%) the K +-evoked release of glutamate from superfused hippocampal synaptosomes in the presence of 100 nM CPA. This effect of CGS 21680 (10 nM), in contrast to the isoproterenol (30 μM) facilitation of glutamate release, was prevented by the protein kinase C inhibitors, chelerythrine (6 μM) and bisindolylmaleimide (1 μM), but not by the protein kinase A inhibitor, H-89 (1 μM). Isoproterenol (30 μM), but not CGS 21680 (10–300 nM), enhanced synaptosomal cAMP levels, indicating that the CGS 21680-induced facilitation of glutamate release involves a cAMP-independent protein kinase C activation. To discard any direct effect of CGS 21680 on adenosine A 1 receptor, we also show that in autoradiography experiments CGS 21680 only displaced the adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentyladenosine ([ 3H]DPCPX, 0.5 nM) with an EC 50 of 1 μM in all brain areas studied and CGS 21680 (30 nM) failed to change the ability of CPA to displace DPCPX (1 nM) binding to CHO cells stably transfected with A 1 receptors. Our results suggest that A 2A receptor agonists facilitate hippocampal synaptic transmission by attenuating the tonic effect of inhibitory presynaptic A 1 receptors located in glutamatergic nerve terminals. This might be a fine-tuning role for adenosine A 2A receptors to allow frequency-dependent plasticity phenomena without compromising the A 1 receptor-mediated neuroprotective role of adenosine.

Protein kinases phosphorylating acidic ribosomal proteins from yeast cells.

Phosphorylation of ribosomal acidic proteins of Saccharomyces cerevisiae is an important mechanism regulating a number of active ribosomes. The key role in the regulatory mechanism is played by specific phosphoprotein kinases and phosphoprotein phosphatases. Three different cAMP-independent protein kinases phosphorylating acidic ribosomal proteins have been identified and characterized. The protein kinase 60S (PK60S), RAP kinase, and casein kinase type 2 (CK2). All three protein kinases phosphorylate serine residues which are localized in the C-terminal end of phosphoproteins. Synthetic peptides were used to determinate the amino acid sequence of phosphoacceptor site for PK60S. Peptide AAEESDDD derived from phosphoproteins YP1 beta/beta' and YP2 alpha turned out to be the best substrate for PK60S. A number of halogenated benzimidazoles and 2-azabenzimidazoles were tested as inhibitors of the three protein kinases. 4,5,6,7-Tetrabromo-2-azabenzimidazole inhibits phosphorylation only of these polypeptides phosphorylated by protein kinase 60S, namely YP1 beta/beta' and YP2 alpha, but not the other, YP1 alpha and YP2 beta phosphorylated by protein kinases RAP and CK2. RAP kinase has been found in an active form in the soluble fraction of S. cerevisiae. The enzyme uses ATP as a phosphate donor and is less sensitive to heparin than casein kinase 2. RAP kinase monophosphorylates the four acidic proteins. The ribosome-bound proteins are a better substrate for the enzyme. Multifunctional CK2 kinase phosphorylate all four acidic proteins. The kinase phosphorylates preferentially serine or threonine residues surrounded by cluster of acidic residues. The enzyme activity is stimulated in vitro by the presence of polylysine and inhibited by heparin.

Maturation-dependent modification of the protein phosphorylation profile of isolated goat sperm plasma membrane.

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca(2+)-calmodulin, phosphatidylserine-diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.

Enzymatic phosphorylation of soy globulins by the protein kinase CK2. Determination of the phosphorylation sites of beta-conglycinin alpha subunit by mass spectrometry.

Beta-conglycinin alpha subunit has been phosphorylated using a cAMP-independent protein kinase (CK2) purified from the yeast Yarrowia lipolytica. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Asp/Glu. Only 0.5 to 1 mol P/mol alpha subunit was incorporated although seven consensus sequences are present. Phosphorylated beta-conglycinin alpha subunit (P-alpha) was digested by trypsin. The resulting peptides were analysed by RP-HPLC coupled to electrospray ionisation mass spectrometry (LC-ESMS). Two phosphopeptides were identified corresponding to 70-89 and 116-127 sequences with Ser 75 and Ser 117 phosphorylated respectively. Ser 75 is one of the predicted phosphorylation sites according to the consensus sequence criteria. Ser 117 is inside a very acidic peptide but does not belong to a previously described consensus sequence.

Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation.

Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.

Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation

Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes. (Hepatology 1996 Dec;24(6):1468-74)

Glucose-induced, cyclic-AMP-independent signalling pathway for activation of neutral trehalase in the fission yeast Schizosaccharomyces pombe

Summary: The addition of glucose to derepressed cells of Schizosaccharomyces pombe provokes a cAMP signal and activation of the cytoplasmic neutral trehalase. This transduction pathway does not require functional RAS protein since RAS1-disrupted cells exhibited a glucose response similar to that shown by control cells. Treatment of activated trehalase by alkaline phosphatase resulted in enzyme deactivation suggesting that trehalase may be modulated in vivo by reversible phosphorylation through cAMP-dependent protein kinase (PKA1). However, the addition of glucose to derepressed growing cells of Schiz. pombe lacking the catalytic subunit of protein kinase A (Δpka1::URA4+ strains) induced stimulation of trehalase as well as phosphorylation of the enzyme protein. This glucose-induced response was absent in PKA1-deficient cells from resting cultures. Addition of exogenous cAMP activated trehalase in normal growing cells but failed to produce any effect on trehalase in PKA1-disrupted growing cells. These results confirm the occurrence of a PKA1-dependent pathway for trehalase activation and imply the existence of another glucose-induced phosphorylation pathway capable of activating trehalase during growth by a distinct, cAMP-independent protein kinase. At least one of the upstream components playing a role in the transduction of this alternative signal is either absent or inactive in cells from stationary phase and sporulated cultures. Cells harbouring the disrupted PKA1 gene responded also to a heat-shock signal by increasing trehalase activity, thus revealing that this enzyme may be a target common to various signalling pathways in the fission yeast.

Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases

The regulation of cardiac muscle glycogen metabolism is not well understood. Previous studies have indicated that heart glycogen synthase is heavily phosphorylated in vivo on multiple sites. Using purified enzymes, we have investigated the effect of phosphorylation of different sites on the activity of rat heart glycogen synthase. A convenient procedure was developed for the purification of rat heart glycogen synthase. The enzyme was phosphorylated by selected kinases, and glycogen synthase activity, extent of phosphorylation, and phosphopeptide maps were analyzed. Rat heart glycogen synthase, purified to apparent homogeneity (M(r) 87,000 on SDS-PAGE), had a specific activity of 18 U/mg protein and had an activity ratio of 0.74 (activity in the absence divided by the activity in the presence of glucose 6-P). cAMP-dependent protein kinase, glycogen synthase kinase 3, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and phosphorylase kinase phosphorylated the enzyme with a concomitant decrease in the activity ratio to values ranging from 0.1 to 0.4. Casein kinase II phosphorylated but did not inactivate glycogen synthase. Six tryptic phosphopeptides, obtained from heart glycogen synthase phosphorylated by the various kinases, were separated by reverse-phase chromatography. The phosphopeptide(s) obtained with each kinase eluted at the same position(s) as corresponding phosphopeptides obtained from rat skeletal muscle glycogen synthase. The study shows that the pattern of phosphorylation and effects on activity are very similar for cardiac and skeletal muscle glycogen synthase. It is suggested that the well known differences in heart and glycogen metabolism may be due to the interplay of kinases and phosphatases which could lead to different phosphorylation and activity states of glycogen synthase.