Biomedical subjects

H LeVine

Publications and source records attributed to H LeVine.

At least 37 records · Page 2Linked to original sources

PubMed1990

Isolation of a novel visual-system-specific arrestin: an in vivo substrate for light-dependent phosphorylation.

Absorption of a photon of light by rhodopsin triggers mechanisms responsible for excitation as well as regulation of the phototransduction cascade. Arrestins are a family of proteins that appear to be responsible for terminating the active state of G-protein-coupled receptors. One of the major substrates of light-dependent phosphorylation in the visual cascade of Drosophila was purified and partially sequenced. The complete primary structure of the protein was determined by isolating the corresponding gene, which revealed it to be a new isoform of arrestin, Arr2. Arr2 is 401 residues in length, and shares 47% sequence identity with the Drosophila Arr1 protein and 42% with human arrestin. We show that the two Drosophila arrestin genes are differentially regulated, and that Arr2 is a specific substrate for a calcium-dependent protein kinase. This is the first demonstration of in vivo regulation of arrestins in a transduction cascade, and provides a new level of modulation in the function of G-protein-coupled receptors.

Amino Acid Sequence↗

PubMed1988

A monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin.

A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca2+ (EC50 = 20 microM) for the interaction. Other divalent cations such as Zn2+, Mn2+, Cd2+, Co2+, and Ni2+ will substitute for Ca2+, while Mg2+ and Ba2+ will not. The antibody reacts with both the alpha- and beta-subunits on Western blots in a similar Ca2+-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of 125I Bolton-Hunter-labeled kinase with unlabeled enzyme. A variety of other proteins including tubulin do not compete for antibody binding. The Mr 30,000 catalytic fragment obtained by proteolysis of either the soluble or the cytoskeletal form of the kinase fails to react with the antibody. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the Kmapp for calmodulin and an increase in the Vmax for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the Mr 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca2+-dependent conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Two types of brain calmodulin-dependent protein kinase II: morphological, biochemical and immunochemical properties.

Two forms of the soluble calmodulin-dependent protein kinase type II can be isolated from rat brain: one oligomeric enzyme complex contains the alpha and beta subunits of the enzyme, whereas the other oligomeric species is comprised of a constant ratio of the subunits of the kinase and tubulin in the presence of several other minor polypeptides. The unassociated enzyme oligomer does not detectably exchange with the tubulin-containing form, and both forms rechromatograph by ion-exchange to their respective positions. In the molecular complex of proteins eluting at high ionic strength, the ratio of kinase subunits to tubulin remains constant throughout sedimentation velocity centrifugation and gel permeation chromatography. Furthermore, a similar complex of proteins is coprecipitated by the anti-kinase monoclonal antibody. Hydrodynamic parameters demonstrate that the tubulin-associated enzyme is larger than the unassociated enzyme, and displays heterodisperse behavior as well. Electron microscopic examination of negatively stained enzyme preparations reveals that the free enzyme constitutes uniform 10-20 nm diameter oligomers in contrast to the tubulin-associated kinase which forms elongated structures with varying morphology. Interestingly, enzyme purified through the calmodulin-Sepharose step can also form 'polymers' featuring ultrastructural similarities to postsynaptic densities and brain microsomal cytoskeletal preparations. We discuss the relevance of these observations to the ability of the type II calmodulin-dependent protein kinase to interact with other polypeptides and to form cytoskeletal structures such as the postsynaptic density.

Animals↗

PubMed1987

Amino acid sequence analysis of the neuronal type II calmodulin-dependent protein kinase by tandem mass spectrometry.

The primary structure of the neuronal Type II calmodulin-dependent protein kinase has been examined by protein sequence analysis and compared to cDNA-derived sequence. Tandem mass spectroscopic analysis was used for the sequence determination. Comparison with published cDNA sequence data for the alpha subunit revealed that the difference between the alpha- and beta-subunits lay in two insertions into the sequence for the alpha-subunit and a short alpha-specific sequence. The N-terminal amino acid of the alpha subunit which is blocked to Edman degradation has been tentatively identified as N-acetyl-alanine.

Amino Acid Sequence↗

PubMed1986

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II.

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.

Animals↗

PubMed1986

Involvement of fodrin-binding proteins in the structure of the neuronal postsynaptic density and regulation by phosphorylation.

Novel polypeptides with Mr values about 140,000 bind fodrin and spectrin and are enriched in the postsynaptic density (PSD) compared to other tissues or subcellular fractions. 125I-fodrin binding to these polypeptides is competed for by unlabeled spectrin. These polypeptides are distinct from ankyrin and its proteolytic fragments and from band 4.1 which also bind fodrin. Phosphorylation of PSDs by the endogenous calmodulin-dependent protein kinase markedly reduces 125I-fodrin binding to the transblotted preparation. Such an event may play a regulatory role in governing protein-protein interactions among elements of the PSD.

Animals↗

PubMed1986

Determination of calmodulin by competitive binding assay.

Calmodulin levels in tissue or cellular extracts can be determined by competition with 125I-calmodulin in a filtration-based direct binding assay. The method is rapid, uses readily available stable components, and possesses a selectivity and sensitivity comparable to that observed with immunoassay and phosphodiesterase activation. This assay provides a tool to readily probe changes in calmodulin levels in cells and tissues as a function of pathophysiologic state.

Animals↗

PubMed1986

Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells.

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.

Aminoacridines↗

PubMed1986

Phorbol ester receptors and protein kinase C in primary neuronal cultures: development and stimulation of endogenous phosphorylation.

Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.

Animals↗

PubMed1985

Early postnatal development of calmodulin-dependent protein kinase II in rat brain.

The postnatal levels of the alpha- (Mr 50,000) and beta- (Mr 58,000/60,000) subunits of type II calmodulin-dependent protein kinase were investigated in cytosolic, cytoskeletal and nuclear subfractions of rat brain. Substantial amounts of the beta-subunit were present neonatally, especially in the cytoplasm; both subunits increased markedly during the subsequent three weeks with alpha-preponderance in the cortex and midbrain. The development of the alpha-subunit was attenuated in the cerebellum and brain stem, resulting in a relative excess of the beta-subunit. Primary neuronal cultures from fetal brain initially displayed low enzyme content, followed by an increase in the levels of the beta- but not alpha-subunit. Regulation of the cellular content of type II calmodulin-dependent protein kinase may be relevant to neuronal differentiation.

Animals↗

PubMed1985

Endogenous dephosphorylation of synaptosomal calmodulin-dependent protein kinase type II.

Calmodulin-dependent protein kinase Type II autophosphorylation in synaptosomes is localized to the cytoskeleton (synaptic junction), while a potent dephosphorylating activity is present in the lipid bilayer. The dephosphorylating activity is operative in intact synaptosomes and in a reconstitution system comprised of the cytoskeletal and Triton X-100 - soluble fractions. Dephosphorylation is inhibited by EDTA and pyrophosphate, but not by EGTA or NaF. The present characterization of endogenous synaptosomal dephosphorylating activity completes the regulatory cycle operating on this enzyme in which phosphorylation of calmodulin-dependent protein kinase type II inhibits its response to Ca+2 and calmodulin.

Calcium↗

PubMed1985

Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange.

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.

Amiloride↗

PubMed1985

Cytoskeletal calmodulin-dependent protein kinase. Characterization, solubilization, and purification from rat brain.

Brain calmodulin-dependent protein kinase was isolated as the major cytoskeletal component of the post-synaptosomal fraction, accounting for about 50% of the total protein. The alpha- and beta-subunits of the enzyme were present in a ratio of about 6:1. The cytoskeletal preparation was also distinguished by the regular presence of tubulin and actin in approximately equimolar amounts to the beta-subunit. Electron microscopic morphology suggested that the enzyme formed a hierarchy of cytoskeletal complexes with an average diameter of 85 nm. These results indicate that cytoskeletal calmodulin-dependent protein kinase may have a structural as well as a catalytic role in neurons. The cytoskeletal enzyme could be solubilized in 8 M urea, displaying similar properties to its cytosolic counterpart. The postsynaptosomal cytoskeleton provides a simplified and well-defined model for the study of the protein-protein interactions involving calmodulin-dependent protein kinase.

Animals↗

PubMed1985

Calmodulin binding to the cytoskeletal neuronal calmodulin-dependent protein kinase is regulated by autophosphorylation.

A brain cytoskeletal preparation that is highly enriched in calmodulin-dependent protein kinase facilitated the study of the binding of 125I-labeled calmodulin to the native enzyme. The binding was specific, saturable, Ca2+-dependent, and inhibited by trifluoperazine. Stoichiometric analysis revealed that the ratio of bound calmodulin to the alpha subunit of the protein kinase was about 1:10 (+/-30%), indicating that in the native state not all of the enzyme subunits were accessible to bind calmodulin. The Kd for the binding reaction was 7 X 10(-9) M and was subject to regulation by divalent cations other than Ca2+, decreasing to 1.7 X 10(-9) M in the presence of 7 mM MgCl2. Activation of the protein kinase in the presence of Ca2+ and calmodulin resulted in marked autophosphorylation of the enzyme subunits. The autophosphorylation was accompanied by a 2-fold decrease in the affinity and number of 125I-labeled calmodulin binding sites. This effect was also reflected by an increase in the apparent Km for Ca2+ from 90 to 200 X 10(-9) M. Thus, enzyme autophosphorylation appears to represent a negative feedback signal, rendering the enzyme less sensitive to subsequent stimulation by physiologic increases in the intracellular Ca2+ concentration. These results help to clarify the mode of neuronal intracellular Ca2+ signaling.

Animals↗

PubMed1984

Protein kinase C: rapid enzyme purification and substrate-dependence of the diacylglycerol effect.

Protein kinase C has been purified by a rapid method resulting in a high-yield, stable enzyme preparation. The catalytic and regulatory properties of this enzyme preparation were characterized employing histone H1 and HMG8, a proteolytic fragment of H1. The enzyme had a lower Km for HMG8, and was stimulated more effectively by diacylglycerol and phorbol esters in the presence of this substrate. Furthermore, these activators markedly increased the Km for HMG8 but not for H1. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate serine residues which are located in different, single tryptic peptides from HMG8.

Animals↗

PubMed1984

Ca2+-calmodulin-dependent protein kinase in neuronal nuclei.

Ca2+-calmodulin-dependent protein kinase represents a major enzyme activity in neuronal nuclei. Most of the protein kinase activity is associated with the nuclear matrix but it can phosphorylate a broad range of substrates which derive from different nuclear compartments. Notable among these substrates is a Mr = 50,000 component of the nuclear matrix and a high mobility group chromatin protein which behaves like HMG17. The high level of activity, age- and tissue-specificity, and sensitivity to low concentrations of calmodulin suggest a role for this Ca2+-calmodulin-dependent protein kinase in regulating neuronal function at the level of the nucleus.

Age Factors↗

PubMed1984

Specificity of a phosphatase for phospholipid, Ca2+-dependent protein kinase-phosphorylated histone H1 resides in the catalytic subunit.

A protein phosphatase from liver which acts preferentially on histone phosphorylated with phospholipid, Ca2+-dependent protein kinase has been purified and the intrinsic specificity determined to reside in the catalytic subunit of the enzyme complex. Comparison with a preparation of pork heart protein phosphatase suggests that this specificity may be a general property of a class of protein phosphatases. Protein kinase C-phosphorylated histone H1 represents an improved substrate for phosphatase detection providing a five to tenfold greater sensitivity than other substrates including cAMP-dependent protein kinase phosphorylated H1.

Animals↗

PubMed1984

Ca2+/calmodulin-dependent protein kinases from the neuronal nuclear matrix and post-synaptic density are structurally related.

A major Ca2+/calmodulin-dependent protein kinase has been isolated in association with the neuronal nuclear matrix. Nuclear matrix preparations contain highly phosphorylated polypeptides with Mr values of 50,000 and 60,000. These polypeptides were further characterized by peptide and phospho peptide mapping, two-dimensional isoelectrofocusing/NaDodSO4/PAGE, and 125I-labeled calmodulin binding. The results indicate that the Mr 50,000 and 60,000 polypeptides of the nuclear matrix closely resemble the alpha and beta subunits, respectively, of the Ca2+/calmodulin-dependent protein kinase of the post-synaptic density. These findings indicate that similar protein kinases mediate the neuronal effects of Ca2+ at the cytosolic, synaptosomal, and nuclear levels.

Animals↗