Expression cloning of an activin receptor, a predicted transmembrane serine kinase.

Activins are involved in the regulation of multiple biological events, ranging from early development to pituitary function. To characterize the cellular mechanisms involved in these processes, cDNAs coding for an activin receptor were cloned from AtT20 mouse corticotropic cells by screening COS cell transfectants for binding of 125I-activin A. The cDNAs code for a protein of 494 amino acids comprising a ligand-binding extracellular domain, a single membrane-spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity. 125I-activin A binds to transfected COS cells with an affinity of 180 pM and can be competed by activin A, activin B, and inhibin A, but not by transforming growth factor beta 1. The kinase domain, but not the extracellular sequence, of the activin receptor is most closely related to the C. elegans daf-1 gene product, a putative transmembrane serine/threonine-specific protein kinase for which the ligand is not known.     

Viral serine/threonine protein kinases

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.     

Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Two distinct domains in Drosophila melanogaster telomeres

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.     

Serine/threonine protein kinases in Drosophila

The study of serine/threonine kinases in Drosophila is coming of age. Recently several kinases have been identified and their role in cell determination has been established. This review discusses these recent findings and describes the potential for genetic analyses of kinase activity and signal transduction.     

Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations.

Mitogen-activated protein kinase (p42mapk) becomes transiently activated after treatment of serum-starved murine Swiss 3T3 cells or EL4 thymocytes with a diversity of mitogens. Similarly, a meiosis-activated protein kinase (p44mpk) becomes stimulated during maturation of sea star oocytes induced by 1-methyladenine. Both p42mapk and p44mpk have been identified as protein-serine/threonine kinases that are activated as a consequence of their phosphorylation. Because homologous protein kinases may play essential roles in both mitogenesis and oogenesis, we have compared in detail the biochemical properties of these two kinases. We find that these kinases are highly related based on their in vitro substrate specificities, sensitivity to inhibitors, and immunological cross-reactivity. However, they differ in apparent molecular weight and can be separated chromatographically, indicating that the two enzymes are distinct. Furthermore, in the course of this investigation, we have identified a 44-kDa protein kinase in mitogen-stimulated Swiss mouse 3T3 cells and EL4 thymocytes that co-purifies with p44mpk and thus appears to be a closer homolog of the sea star enzyme. Analysis of these protein kinases clarifies the relationships between a set of tyrosine-phosphorylated 41-45-kDa proteins present in mitogen-stimulated cells (Martinez, R., Nakamura., K. D., and Weber, M. J. (1982) Mol. Cell. Biol. 2, 653-655; Cooper, J. A., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37), two myelin basic protein kinases identified in epidermal growth factor-treated Swiss mouse 3T3 cells (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265, 11487-11494), and p42mapk. Our work points to the existence of a group of related serine/threonine protein kinases, regulated by tyrosine phosphorylation and functioning at different stages of the cell cycle.     

Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors.

We have cloned ActR-IIB, which encodes four new activin receptor isoforms belonging to the protein serine/threonine kinase receptor family. Two of the ActR-IIB isoforms have higher affinity for activin A than the previously cloned activin receptor and differ from each other by the inclusion of an alternatively spliced segment in the cytoplasmic juxtamembrane region. A second alternative splicing event generates two additional receptor isoforms that lack a proline cluster in the external juxtamembrane region and have lower affinity for activin A. All isoforms bind inhibin A with low affinity. Thus, the repertoire of activin receptors includes species that differ in ligand binding affinity, cytoplasmic domain structure, or both. This receptor heterogeneity might underlie the sharply different responses that activin can elicit in a dose- or cell-specific manner.     

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.     

Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.     

Properties of an alpha-bungarotoxin-binding cholinergic nicotinic receptor from Drosophila melanogaster.

alpha-[125I]Bungarotoxin specifically binds to homogenates of Drosophila melanogaster head at levels of 0.3-0.8 pmol/mg protein. The dissociation constant calculated from rates of association and dissociation of toxin.receptor complex, is 0.6.10(-9) M. Ca2+, and to a lesser extent Na+, inhibit the reaction. alpha-[125I]Bungarotoxin binding is inhibited by low concentrations of unlabelled toxin, nicotinic ligands and eserine, but not by low concentrations of muscarinic ligands, decamethonium or an organophosphate. The receptor is membrane bound and can be partially released into 100 000 X g supernatant by combination of 1 M NaCl and 1% Triton X-100. Most of the activity in the supernatant sediments after further centrifugation at 200 000 X g for 2 h. Toxin binding sites are distinct from acetylcholinesterase molecules as revealed by pharmacological, biochemical and genetic techniques. The gene for the toxin-binding nicotinic receptor in Drosophila is apparently not located adjacent to the gene for acetylcholinesterase.     

Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases

Fluorescent chemosensors of protein kinase activity provide a continuous, high-throughput sensing format for the study of the roles of these enzymes, which are crucial for regulating cellular function. Specifically, chemosensors using the nonnatural amino acid, Sox, and physiological Mg(2+) levels report phosphorylation with dramatic fluorescence changes that are amenable to real-time and high-throughput analysis. In this article, we report 15 probes for a total of six distinct serine/threonine kinases with large fluorescence increases and good reactivity toward the target kinase. The sensing mechanism is detailed, and the optimal sensing motif is determined. These versatile and powerful sensors provide tools for researchers studying the roles of the targeted kinases in signal transduction, and the design principles provide guidelines for the generation of future fluorescent chemosensors for any serine/threonine kinase.     

Phosphorylation of atrial natriuretic factor R1 receptor by serine/threonine protein kinases: evidences for receptor regulation

The 130 kDa atrial natriuretic factor receptor (ANF-R₁) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99–126) and there is no detectable intrinsic kinase activity associated with the ANF-R₁ receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for ¹²⁵I-ANF. However, we have demonstrated a significant ³²P incorporation in the ANF-R₁ receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-R₁ receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension.Abbreviations ANF Atrial Natriuretic Factor - ANF-R₁ Atrial Natriuretic Factor Receptor, subtype 1 - ATP Adenosine Triphosphate - CaCl₂ Calcium Chloride - cAMP Adenosine cyclic 3,5-Monophosphate acid - cGMP Guanosine cyclic 35-Monophosphate acid - EDC 1-Ethyl-3-[3-Dimethylaminopropyl] Carbodiimide - EDTA Ethylenediaminetetraacetic Acid - GTP Guanosine Triphosphate - IBMX 3-isobutyl-1-methylxanthine - kDa Kilodaltons - MgCl₂ Magnesium Chloride - MgAC Magnesium Acetate - NaCl Sodium Chloride - PAGE Polyacrylamide Gel Electrophoresis - PKA cAMP-dependent protein kinase - PKG cGMP-dependent Protein Kinase - PKC Calcium/Phospholipid-dependent Protein Kinase - RIA Radioimmunoassay - SDS Sodium Dodecyl Sulfate - SHR Spontaneously Hypertensive Rat - Tris HCl Tris (Hydroxymethyl) aminomethane Hydrochloride - TPA 12-O-Tetradecanoyl-Phorbol-13-Acetate