Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken.

The crystal structures of the catalytic fragment of chicken poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] with and without a nicotinamide-analogue inhibitor have been elucidated. Because this enzyme is involved in the regulation of DNA repair, its inhibitors are of interest for cancer therapy. The inhibitor shows the nicotinamide site and also suggests the adenosine site. The enzyme is structurally related to bacterial ADP-ribosylating toxins but contains an additional alpha-helical domain that is suggested to relay the activation signal issued on binding to damaged DNA.

Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies.

Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick-synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG.

Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta.

Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors.

Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli.

Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

Expression of PTPH1, a rat protein tyrosine phosphatase, is restricted to the derivatives of a specific diencephalic segment.

Studies to date have identified only a few proteins that are expressed in a segment-specific manner within the mammalian brain. Here we report that a nonreceptor protein tyrosine phosphatase, PTPH1, is selectively expressed in the adult thalamus. Expression of PTPH1 mRNA is detected in most, but not all, thalamic nuclei. Nuclei that are derived embryonically from the dorsal thalamus and project to the neocortex express this gene, whereas those derived from the ventral thalamus do not. PTPH1 mRNA expression is also restricted to the dorsal thalamus during development and, thus, can serve as a specific marker for the dorsal thalamic nuclei. Since the subcellular localization of PTPH1 protein is not known, its functional role is not clear. However, the restriction of its expression to the thalamic nuclei that have thalamocortical connections suggests that PTPH1 may play a role in the maintenance of these connections or in determining the physiological properties of thalamic relay nuclei.

Emergent spindle oscillations and intermittent burst firing in a thalamic model: specific neuronal mechanisms.

The rhythmogenesis of 10-Hz sleep spindles is studied in a large-scale thalamic network model with two cell populations: the excitatory thalamocortical (TC) relay neurons and the inhibitory nucleus reticularis thalami (RE) neurons. Spindle-like bursting oscillations emerge naturally from reciprocal interactions between TC and RE neurons. We find that the network oscillations can be synchronized coherently, even though the RE-TC connections are random and sparse, and even though individual neurons fire rebound bursts intermittently in time. When the fast gamma-aminobutyrate type A synaptic inhibition is blocked, synchronous slow oscillations resembling absence seizures are observed. Near-maximal network synchrony is established with even modest convergence in the RE-to-TC projection (as few as 5-10 RE inputs per TC cell suffice). The hyperpolarization-activated cation current (Ih) is found to provide a cellular basis for the intermittency of rebound bursting that is commonly observed in TC neurons during spindles. Such synchronous oscillations with intermittency can be maintained only with a significant degree of convergence for the TC-to-RE projection.

Three-dimensional scroll waves of cAMP could direct cell movement and gene expression in Dictyostelium slugs.

Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.