Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase

The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular ATP (adenosine triphosphate) and AMP concentrations. Here, we report crystal structures at 2.9 and 2.6 A resolution for ATP- and AMP-bound forms of a core alphabetagamma adenylate-binding domain from the fission yeast AMPK homolog. ATP and AMP bind competitively to a single site in the gamma subunit, with their respective phosphate groups positioned near function-impairing mutants. Unexpectedly, ATP binds without counterions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.     

A yeast gene that is essential for release from glucose repression encodes a protein kinase

The SNF1 gene plays a central role in carbon catabolite repression in the yeast Saccharomyces cerevisiae, namely that SNF1 function is required for expression of glucose-repressible genes. The nucleotide sequence of the cloned SNF1 gene was determined, and the predicted amino acid sequence shows that SNF1 encodes a 72,040-dalton polypeptide that has significant homology to the conserved catalytic domain of mammalian protein kinases. Specific antisera were prepared and used to identify the SNF1 protein. The protein was shown to transfer phosphate from adenosine triphosphate to serine and threonine residues in an in vitro autophosphorylation reaction. These findings indicate that SNF1 encodes a protein kinase and suggest that protein phosphorylation plays a critical role in regulation by carbon catabolite repression in eukaryotic cells.     

Repair of damaged DNA in a eucaryotic cell: Tetrahymena pyriformis

Damage induced by ultraviolet light or x-rays to the DNA of a eucaryotic organism, Tetrahymena pyriformis, is repaired by a process similar to the repair system present in bacteria. This repair process, which involves defect excision and subsequent resynthesis of the damaged section of DNA, occurs in the dark. Photoreactivation of damage induced by ultraviolet light is also indicated by a reduction in observed repair synthesis. An improved method for detecting repair synthesis is described. Repair synthesis is measured in parental DNA strands isolated from cultures that have undergone normal DNA replication after the repair process.     

TRP-PLIK, a bifunctional protein with kinase and ion channel activities

We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing alpha-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function.     

Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase

The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication. A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells. RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity. The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication. These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.     

Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.     

Phosphorylation of muscle membranes: identification of a membrane-bound protein kinase

A membrane-bound protein kinase occurs in membranes derived from rat skeletal muscle and appears limited to a surface membrane fraction. The enzyme is magnesium dependent, is only minimally stimulated by cyclic nucleotides, and phosphorylates serine and to a lesser extent threonine residues of three membrane proteins with molecular weights of less than 30,000.     

Expression and properties of two types of protein kinase C: alternative splicing from a single gene

Two complementary DNA's, encoding the complete sequences of 671 and 673 amino acids for subspecies of rat brain protein kinase C, were expressed in COS 7 cells. The complementary DNA sequence analysis predicted that the two enzymes are derived from different ways of splicing and differ from each other only in the short ranges of their carboxyl-terminal regions. Both enzymes showed typical characteristics of protein kinase C that responded to Ca2+, phospholipid, and diacylglycerol. The enzymes showed practically identical physical and kinetic properties and were indistinguishable from one of the several subspecies of protein kinase C that occurs in rat brain but not in untransfected COS 7 cells. Partial analysis of the genomic structure confirmed that these two subspecies of protein kinase C resulted indeed from alternative splicing of a single gene.     

Gene product of v-fgr onc: hybrid protein containing a portion of actin and a tyrosine-specific protein kinase

The nucleotide sequence of the region of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encoding its primary translation product, p70gag-fgr, has been determined. From the nucleotide sequence, the amino acid sequence of this transforming protein was deduced. Computer analysis indicates that a portion of P70gag-fgr has extensive amino acid sequence homology with actin, a eukaryotic cytoskeletal protein. A second region of P70gag-fgr is closely related to the tyrosine-specific kinase gene family. Thus, the v-fgr oncogene appears to have arisen as a result of recombinational events involving two distinct cellular genes, one coding for a structural protein and the other for a protein kinase.     

内蒙古螺旋藻HSP90基因的克隆及三亲接合表达载体构建

内蒙古螺旋藻是一种原产于内蒙古碱湖,品质优良、中温适应型的螺旋藻品种,但因发现较晚,生长环境特殊等原因,对其分子遗传学等基础研究相对滞后。提高其耐热性能够扩大养殖范围,降低养殖成本,具有很大的应用价值。研究表明热激蛋白在藻类抗高温胁迫中起着重要作用。本研究采用PCR技术得到内蒙古螺旋藻HSP90基因序列,并对基因及其编码蛋白序列进行了初步分析;同时,根据蓝藻遗传转化需要,构建了三亲接合转移的穿梭表达载体,该载体起始密码子ATG与目的基因HSP90相距6 bp,含有进行三亲结合转移所需的蓝藻复制起始区、Bom基因、启动子(BetaP)、氯霉素抗性基因(Cm)及目的基因HSP90。研究结果为构建耐热内蒙古螺旋藻转基因新品系等奠定了基础。     

Partial reversion in yeast: genetic evidence for a new type of bifunctional protein

Two kinds of phenotypic expression in purine biosynthesis result from recessive mutation to adel2 in baker's yeast. The mutants are adenine-specific, blocked in the conversion of inosine 5'-phosphate to adenylosuccinic acid; their response to inhibition of pathway activity by adenine is considerably reduced. Allelic partial reversions can restore prototrophy without correcting the regulatory defect imparted by the primary mutation. The separation of the two properties of the locus by allelic mutation supports the hypothesis that the locus specifies a protein of two independent functions, enzymatic and regulatory.     

Cloning of complementary DNA for GAP-43, a neuronal growth-related protein

GAP-43 is one of a small subset of cellular proteins selectively transported by a neuron to its terminals. Its enrichment in growth cones and its increased levels in developing or regenerating neurons suggest that it has an important role in neurite growth. A complementary DNA (cDNA) that encodes rat GAP-43 has been isolated to study its structural characteristics and regulation. The predicted molecular size is 24 kilodaltons, although its migration in SDS-polyacrylamide gels is anomalously retarded. Expression of GAP-43 is limited to the nervous system, where its levels are highest during periods of neurite outgrowth. Nerve growth factor or adenosine 3',5'-monophosphate induction of neurites from PC12 cells is accompanied by increased GAP-43 expression. GAP-43 RNA is easily detectable, although at diminished levels, in the adult rat nervous system. This regulation of GAP-43 is concordant with a role in growth-related processes of the neuron, processes that may continue in the mature animal.     

Potentiation of bleomycin lethality by anticalmodulin drugs: a role for calmodulin in DNA repair

Treatment of exponentially growing Chinese hamster ovary cells with bleomycin causes a dose-dependent decrease in cell survival due to DNA damage. This lethal effect can be potentiated by the addition of a nonlethal dose of the anticalmodulin drug N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide ( W13 ) but not its inactive analog N-(4-aminobutyl)-2-naphthalenesulfonamide ( W12 ). By preventing the repair of damaged DNA, W13 also inhibits recovery from potentially lethal damage induced by bleomycin. These data suggest a role for calmodulin in the DNA repair pathway.     

Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.     

Activators of protein kinase C induce dissociation of CD4, but not CD8, from p56lck

The CD4 and CD8 T cell receptor accessory molecules can both be isolated from T lymphocytes in association with p56lck, a membrane-associated, cytoplasmic tyrosine protein kinase that is expressed exclusively in lymphoid cells. The enzymatic activity of p56lck may therefore be regulated by CD4 and CD8 and be important in antigen-induced T cell activation. Exposure of human T cells and some mouse T cells to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, caused the dissociation of p56lck and CD4. Activation of protein kinase C may therefore interrupt regulation of p56lck by CD4 and alter the ability of p56lck to interact with polypeptide substrates. In contrast, exposure of cells to TPA did not cause dissociation of p56lck and CD8. Regulation of p56lck by CD4 may therefore differ from regulation by CD8.     

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.     

Reaction of the antitumor antibiotic CC-1065 with DNA: structure of a DNA adduct with DNA sequence specificity

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.     

Overexpression of a maize SNF-related protein kinase gene,ZmSnRK2.11, reduces salt and drought tolerance in Arabidopsis

Sucrose non-fermenting-1 related protein kinase 2(Sn RK2) is a unique family of protein kinases associated with abiotic stress signal transduction in plants. In this study, a maize Sn RK2 gene Zm Sn RK2.11 was cloned and characterized. The results showed that Zm Sn RK2.11 is up-regulated by high-salinity and dehydration treatment, and it is expressed mainly in maize mature leaf. A transient expression assay using onion epidermal cells revealed that ZmS nR K2.11-GFP fusion proteins are localized to both the nucleus and cytoplasm. Overexpressing-Zm Sn RK2.11 in Arabidopsis resulted in salt and drought sensitivity phenotypes that exhibited an increased rate of water loss, reduced relative water content, delayed stoma closure, accumulated less free proline content and increased malondialdehyde(MDA) content relative to the phenotypes observed in wild-type(WT) control. Furthermore, overexpression of Zm Sn RK2.11 up-regulated the expression of the genes ABI1 and ABI2 and decreased the expression of DREB2 A and P5CS1. Taken together, our results suggest that Zm Sn RK2.11 is a possible negative regulator involved in the salt and drought stress signal transduction pathways in plants.