Ecto-protein kinase activity on the external surface of neural cells

ATP is secreted in association with neurotransmitters at certain synapses and neuromuscular junctions. Extracellular ATP is known to exert potent effects on the activity of cells in the nervous system, where it can act as a neurotransmitter or as a modulator regulating the activity of other neurohormones. We have suggested that such modulation may involve the activity of extracellular protein phosphorylation systems. It is well known that intracellular protein kinases are important in the regulation of various neuronal functions, but protein kinases which use extracellular ATP to phosphorylate proteins localized at the external surface of the plasma membrane (ecto-protein kinases) have not been demonstrated in neuronal cells. Here we present direct evidence for the existence of an ecto-protein kinase and demonstrate endogenous substrates for its activity at the surface of intact neural cells. The phosphorylation of one of these surface proteins is selectively stimulated during cell depolarization. In addition, neuronal cell adhesion molecules (N-CAMs) appear to be among the substrates of ecto-protein kinase activity. These results suggest a role for surface protein phosphorylation in regulating specific functions of developing and mature neurones.     

Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation

Tyrosine-specific protein phosphorylation has been implicated in the regulation of cell transformation and proliferation. However, recent studies have shown that the expression of protein tyrosine kinases in adult brain is very high, suggesting that tyrosine-specific protein phosphorylation may also have a role in the regulation of neuronal function. Although a number of substrate proteins are phosphorylated on tyrosine residues, the functional alteration of proteins by tyrosine phosphorylation has previously been convincingly demonstrated only for protein tyrosine kinases. The nicotinic acetylcholine receptor, a neurotransmitter-gated ion channel, is phosphorylated by a protein tyrosine kinase in post-synaptic membranes in vitro and in vivo. We demonstrate here that this tyrosine phosphorylation increases the rate of the rapid phase of desensitization of the nicotinic receptor, as measured by single channel recording of purified nicotinic acetylcholine receptor, when reconstituted in lipid vesicles. These data provide direct evidence for the regulation of ion channel properties by tyrosine phosphorylation. The results, which demonstrate a functional role of tyrosine phosphorylation in the nervous system, suggest a widespread role for tyrosine phosphorylation in neuronal signal transduction.     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K+ channels of Aplysia sensory neurones in cell-free membrane patches

Selected actions of neurotransmitters and hormones on ion channels in nerve and muscle cells are now thought to be mediated by cyclic AMP-dependent protein phosphorylation. Although the cyclic AMP-dependent protein kinase (cAMP-PK) affects the cellular properties of several neurones, its mode of action at the single-channel level has not been characterized. In addition, little is known about the identity or subcellular localization of the phosphoproteins that control channel activity and, in particular, whether the critical substrate proteins are cytoplasmic or membrane-associated. In Aplysia sensory neurones, serotonin produces a slow modulatory synaptic potential mediated by cAMP-PK that contributes to presynaptic facilitation and behavioural sensitization. Previously, we have found that serotonin acts on cell-attached membrane patches to produce prolonged all-or-none closures of a specific class of K+ channels (S channels) whose gating is weakly dependent on voltage and independent of intracellular calcium. We demonstrate here that in cell-free membrane patches from Aplysia sensory neurones, the purified catalytic subunit of cAMP-PK produces all-or-none closures of the S channel, simulating most (but not all) aspects of the action of serotonin on cell-attached patches. This result suggests that protein kinase acts on the internal surface of the membrane to phosphorylate either the channel itself or a membrane-associated protein that regulates channel activity.     

A novel viral oncogene with structural similarity to phospholipase C

Numerous oncogenes have been isolated from acutely transforming retroviruses. To date, the products of these viral oncogenes have been protein kinases, nuclear proteins, growth factors, or GTP-binding proteins. We have cloned the previously uncharacterized avian sarcoma virus CT10 and sequenced its genome. This virus encodes a protein, p47gag-crk, that has blocks of sequence similarity to the amino-terminal, non-catalytic region of the non-receptor class of tyrosine kinases. In addition, the structure of p47gag-crk has striking similarity to a 180-amino acid region of bovine brain phospholipase C. Biochemical data suggest that p47gag-crk activates one or several endogenous tyrosine kinases.     

A gene deleted in Kallmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules

Kallmann's syndrome (clinically characterized by hypogonadotropic hypogonadism and inability to smell) is caused by a defect in the migration of olfactory neurons, and neurons producing hypothalamic gonadotropin-releasing hormone. A gene has now been isolated from the critical region on Xp22.3 to which the syndrome locus has been assigned: this gene escapes X inactivation, has a homologue on the Y chromosome, and shows an unusual pattern of conservation across species. The predicted protein has significant similarities with proteins involved in neural cell adhesion and axonal pathfinding, as well as with protein kinases and phosphatases, which suggests that this gene could have a specific role in neuronal migration.     

FMRFamide reverses protein phosphorylation produced by 5-HT and cAMP in Aplysia sensory neurons

Neurotransmitter can modulate neuronal activity through a variety of second messengers that act on ion channels and other substrate proteins. The most commonly described effector mechanism for second messengers in neurons depends on protein phosphorylation mediated by one of three sets of kinases: the cyclic AMP-dependent protein kinases, the Ca2+-calmodulin-dependent protein kinases, and the Ca2+-phospholipid-dependent protein kinases. In addition, some neurotransmitters and second messengers can also inhibit protein phosphorylation by lowering cAMP levels (either by inhibiting adenylyl cyclase or activating phosphodiesterases). This raises the question: can neurotransmitters also modulate neuronal activity by decreasing protein phosphorylation that is independent of cAMP? Various biochemical experiments show that a decrease in protein phosphorylation can arise through activation of a phosphatase or inhibition of kinases. In none of these cases, however, is the physiological role for the decrease in protein phosphorylation known. Here we report that in Aplysia sensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels of protein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides the cAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), and concomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT. These findings indicate that a receptor-mediated decrease in protein phosphorylation may play an important part in the modulation of neurotransmitter release.     

Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.     

A chemical switch for inhibitor-sensitive alleles of any protein kinase

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.     

Boron-based Drug Design for Cancer Therapy

1 Results Selective inhibition of protein tyrosine kinases is gaining importance as an effective therapeutic approach for the treatment of a wide range of human cancers.The epidermal growth factor receptor (EGFR) protein tyrosine kinase is one of the important kinases that play a fundamental role in cell growth signal pathways.We focused on the 4-anilinoquinazoline framework,which is observed in both compounds as a common structure.A boron atom has a vacant orbital and interconverts with ease between th...     

Odorant-sensitive adenylate cyclase may mediate olfactory reception

The mechanism of the sense of smell has long been a subject for theory and speculation. More recently, the notion of odorant recognition by stereospecific protein receptors has gained wide acceptance, but the receptor molecules remained elusive. The recognition molecules are believed to be quite diverse, which would partly explain the unusual difficulties encountered in their isolation by conventional ligand-binding techniques. An alternative approach would be to probe the receptors through transductory components that may be common to all receptor types. Here we report the identification of one such transductory molecular component. This is an odorant-sensitive adenylate cyclase, present in very large concentrations in isolated dendritic membranes of olfactory sensory neurones. Odorant activation of the enzyme is ligand and tissue specific, and occurs only in the presence of GTP, suggesting the involvement of receptor(s) coupled to a guanine nucleotide binding protein (G-protein). The olfactory G-protein is independently identified by labelling with bacterial toxins, and found to be similar to stimulatory G-proteins in other systems. Our results suggest a role for cyclic nucleotides in olfactory transduction, and point to a molecular analogy between olfaction and visual, hormone and neurotransmitter reception. Most importantly, the present findings reveal new ways to identify and isolate olfactory receptor proteins.     

Retinal ganglion cells lose response to laminin with maturation

The decisive role played by adhesive interactions between neuronal processes and the culture substrate in determining the form and extent of neurite outgrowth in vitro has greatly influenced ideas about the mechanisms of axonal growth and guidance in the vertebrate nervous system. These studies have also helped to identify adhesive molecules that might be involved in guiding axonal growth in vivo. One candidate molecule is laminin, a major glycoprotein of basal laminae which has been shown to induce a wide variety of embryonic neurones to extend neurites in culture. Moreover, laminin is found in large amounts in injured nerves that can successfully regenerate but is absent from nerves where regeneration fails. However, it is unclear to what extent the mechanisms that regulate axonal regeneration also operate in the embryo when axon outgrowth is initiated. Here we have examined the substrate requirements for neurite outgrowth in vitro by chick embryo retinal ganglion cells, the only cells in the retina to send axons to the brain. We show that while retinal ganglion cells from embryonic day 6 (E6) chicks extend profuse neurites on laminin, those from E11 do not, although they retain the ability to extend neurites on astrocytes via a laminin-independent mechanism. This represents the first evidence that central nervous system neurones may undergo a change in their substrate requirements for neurite outgrowth as they mature.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Regulation of glutamate receptor binding by the cytoskeletal protein fodrin

The erythrocyte cytoskeleton, which consists primarily of a meshwork of spectrin and actin, controls cell shape and the disposition of proteins within the membrane. Proteins similar to spectrin have recently been found in diverse cells and tissues, and it is possible that they mediate the capping of cell-surface receptors, although this has not been demonstrated directly. In neurones, the spectrin-like protein fodrin lines the cortical cytoplasm and may link actin filaments to the membrane. Fodrin has been hypothesized to regulate the number of receptor binding sites on neuronal membranes for the putative neurotransmitter L-glutamate. Micromolar calcium concentrations activate the thiol protease calpain I, induce fodrin degradation and more than double the density of glutamate binding sites; these effects are all blocked by thiol protease inhibitors. We have now used specific antibodies to examine further the role of fodrin proteolysis in regulating glutamate receptors. We report that fodrin antibodies block the fodrin degradation and increase in glutamate binding normally induced by calcium, and so provide direct evidence for control of membrane receptors by a non-erythroid spectrin.     

Retrograde axonal transport of target tissue-derived macromolecules

Neurones depend on contact with their target tissues for survival and subsequent development. The protein, nerve growth factor (NGF), can be selectively taken up by sympathetic nerve terminals and reaches the neuronal perikaryon by a process of retrograde intra-axonal transport, suggesting that its role in vivo is to act as a target tissue-derived trophic factor. The development of the neurones of the chick ciliary ganglion requires the presence of structures derived from the optic cup. Several studies in vitro have shown that media conditioned by non-neuronal cells contain factors that result in the survival of neurones from ciliary ganglia. In particular, chick embryo iris, ciliary body and choroid contained large amounts of these factors indicating the presence of a target tissue-derived trophic factor for the cholinergic ciliary ganglion. This study demonstrates that neurones of the ciliary ganglion accumulate, by retrograde intra-axonal transport, proteins synthesized and released by optic tissues in culture.     

CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth nor promotes DNA replication in the absence of CDK activity, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.     

Modulation of brain polyphosphoinositide metabolism by ACTH-sensitive protein phosphorylation

Phosphorylation of membrane components is thought to be an important process in membrane function. Phosphorylated proteins and a special class of phospholipids, the (poly)phosphoinositides (poly PI), are implicated in the regulation of membrane permeability and synaptic transmission in neurones. For many years, protein phosphorylation and poly PI metabolism have been studied in parallel without knowledge of their possible interaction. We report here that the ACTH-sensitive protein kinase/B-50 protein complex which we recently isolated in soluble form from rat brain synaptosomal plasma membranes has lipid phosphorylating activity. Exogenously added phosphatidylinositol 4-phosphate (DPI) is phosphorylated to phosphatidylinositol 4,5-diphosphate (TPI), and this DPI-kinase activity is dependent on the state of phosphorylation of the protein kinase/B-50 protein complex. The results imply that phosphorylation of protein may affect the metabolism of (poly) PI in brain cell membranes.