Jack Erlichman

Localization and characterization of the binding site for the regulatory subunit of type II cAMP-dependent protein kinase on MAP2

Microtubule-associated protein 2 (MAP2) binds, and is a substrate for, type II cAMP-dependent protein kinase. The structural domain in MAP2 that binds the regulatory subunit (RII) of protein kinase II was identified by expressing fragments of a human MAP2 cDNA in E. coli using the pATH11 vector. Fusion proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The filters were probed with purified bovine heart or brain RII, anti-RII monoclonal antibodies, and 125I-labeled protein A. Binding of RII was localized to a 31 amino acid sequence near the N-terminus of the MAP2 molecule. Fusion proteins containing this fragment bound both heart and brain RIIs in a concentration-dependent manner, but bound heart RII with a higher apparent affinity than brain RII. The amino acid sequence of this fragment (DRETAEEVSARIVQVVTAEAVAVLKGEQEKE) is totally conserved between human and mouse MAP2, suggesting an important role for the RII binding site of MAP2 in neuronal function.

[44] Cyclic AMP-dependent protein kinase from bovine heart muscle

Publisher Summary Cyclic AMP (cAMP)-dependent protein kinase catalyzes the transfer of the γ-phosphate of ATP to serine or threonine hydroxyl groups in various protein substrates. The addition of cAMP greatly enhances the velocity of the phosphotransferase reaction. The activity of the enzyme is determined by measuring the amount of 32 P transferred from [γ -32 P] ATP to protamine or histone. The labeled protein substrate is separated from the assay mixture by precipitation with trichloroacetic acid and collected on glass fiber filters. Assays are carried out in the presence and absence of cAMP to evaluate the activation of the enzyme by the cyclic nucleotide. One unit of protein kinase activity is defined as that amount of enzyme necessary to catalyze the transfer of 1 nmole of 32 P from [γ -32 P] ATP to protamine per minute at 30°C. A unit of binding activity is equivalent to the binding of 1 nmole of cAMP. Specific activity is expressed as units per milligram of protein.

The development of hormone sensitivity by the adenyl cyclase of the tadpole erythrocyte

Abstract Catecholamines have previously been shown to stimulate adenyl cyclase activity in hemolyzates of frog erythrocytes but not in hemolyzates prepared from the erythrocytes of premetamorphic tadpoles. This observation has now been confirmed in intact erythrocytes from the frog and tadpole as well as in partially purified, particulate adenyl cyclases prepared from these cells. The development of sensitivity to stimulation by catecholamines was studied in hemolyzates prepared from tadpoles undergoing either natural or thyroxin-induced metamorphosis. Sensitivity was first detected in erythrocytes from tadpoles which had developed front legs and were undergoing the process of tail resorption. Acrylamide gel electrophoresis of a hemolyzate from this stage of development revealed a new band of hemoglobin which corresponded to the position of adult frog hemoglobin. The sensitivity of tadpole erythrocyte adenyl cyclase to activation by β-adrenergic compounds increased during the period of tail resorption and approached that of the adult frog upon completion of metamorphosis.

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RIIβ of type II cAMP-dependent protein kinase

Abstract The promoter and exon 1 of the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5′-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RIIβ 5′-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp −291 −121 . An enhancer element was located between bp −1426 −1018 .

Cyclic AMP binding to the amphibian oocyte plasma membrane: possible interrealtionship between meiotic arrest and membrane fluidity

Cyclic AMP, which maintains the vertebrate oocyte in prophase arrest under physiological conditions, exhibits specific and saturable binding to the cytoplasmic face of the prophase-arrested Rana pipiens oocyte plasma membrane. Scatchard type analyses of [3H]cAMP binding to isolated plasma membranes indicate a single class of binding sites with a Kd = 19.3 +/- 7.0 nM at cAMP concentrations below 10(-6) M and additional low affinity site(s) and/or non-specific binding at concentrations above 10(-6) M. Photoaffinity labeling of prophase oocyte plasma membranes with [32P]-8-N3cAMP demonstrates cAMP/cGMP-displacable binding of 8-N3[32P]cAMP to a 100-110 kDa peptide doublet. Plasma membrane fluidity was monitored by electron spin resonance in isolated plasma-vitelline membranes using a 5-doxyl stearic acid probe. Exogenous dibutyryl cAMP (dbcAMP) produces an increase in membrane fluidity within minutes and blocks and/or reverses the progesterone-induced decrease in plasma membrane fluidity. The dbcAMP concentration that produced half-maximal fluidity increase (10 microM) corresponds to the half-maximal inhibiting dose of dbcAMP for progesterone induction of meiosis. Cholera toxin, which elevates intracellular cAMP and blocks meiosis, also increases membrane fluidity and inhibits progesterone-induced decrease in membrane fluidity. Elevated levels of intracellular cAMP thus appear to maintain meiotic arrest by binding to specific plasma membrane site(s) and maintaining the plasma membrane in a relatively fluid state. The progesterone-induced fall in intracellular cAMP first reported in Rana thus appears to be responsible for the progesterone-induced increase in membrane fluidity and further suggests that the change in membrane order is essential for the resumption of the meiotic divisions.

[18] Reversible autophosphorylation of type II cAMP-dependent protein kinase: Distinction between intramolecular and intermolecular reactions

Publisher Summary This chapter discusses the distinction between intramolecular and intermolecular reactions. Many proteins are subject to reversible covalent modification by phosphorylation and it is widely recognized that this modification is a critical component of the physiological response of mammalian cells to specific stimuli. The cAMP-dependent protein kinases (PK) catalyze the initial phosphorylation reaction in cAMP-mediated pathways. In most tissues and cells there are two classes of cAMP-dependent protein kinases, designated types I and II by their order of elution from diethylaminoethyl (DEAE)-cellulose. The chapter describes techniques to measure the reversible phosphorylation of the exchangeable site on type II R and distinguish between intramolecular and intermolecular phosphorylation of this site.

Activation of cyclic AMP-dependent protein kinase isoenzymes: Studies using specific antisera

A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nM for protein kinase I and 80 nM for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [gamma-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.

Molecular Characterization of Cyclic AMP-Dependent Protein Kinases Derived from Bovine Heart and Human Erythrocytes

The soluble cyclic AMP-dependent protein kinase of bovine heart has been purified to homogeneity. It is an asymmetric protein with a molecular weight of 174,000 and is composed of two globular catalytic subunits (molecular weight, 38,000) and an asymmetric cyclic nucleotide-binding protein (molecular weight, 98,000). The cyclic AMP-binding protein consists of two polypeptide chains of equal size and is able to serve as substrate for a protein kinase-catalyzed phosphotransferase reaction. The physiological significance of this “autophosphorylation” of protein kinase has not been established. The protein kinase activity associated with the plasma membrane of the human erythrocyte has many properties in common with soluble protein kinases including the ability to be dissociated into cyclic AMP-independent kinase and cyclic AMP-binding activities. Membrane-bound protein kinase catalyzes the phosphorylation of three endogenous membrane proteins, two of them in a cyclic nucleotide-dependent fashion. Both the cyclic AMP-binding and phosphotransferase activities of the human erythrocyte membrane appear to be localized on the inner cytoplasmic surface of the membrane.

cAMP-dependent protein kinases in the rat testis : regulatory and catalytic subunit associations

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.