Ras2 and Ras1 protein phosphorylation in Saccharomyces cerevisiae

This work describes the phosphorylation of Saccharomyces cerevisiae Ras proteins and explores the physiological role of the phosphorylation of Ras2 protein. Proteins expressed from activated alleles of RAS were less stable and less phosphorylated than proteins from cells expressing wild-type alleles of RAS. This difference in phosphorylation level did not result from increased signaling through the Ras-cAMP pathway or reflect the primarily GTP-bound nature of activated forms of Ras protein per se. In addition, phosphorylation of Ras protein was not dependent on proper localization of the Ras2 protein to the plasma membrane nor on the interaction of Ras2p with its exchange factor, Cdc25p. The preferred phosphorylation site on Ras2 protein was identified as serine 214. This site, when mutated to alanine, led to promiscuous phosphorylation of Ras2 protein on nearby serine residues. A decrease in phosphorylation may lead to a decrease in signaling through the Ras-cAMP pathway.     

Interaction of a novel GDP exchange inhibitor with the Ras protein

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.     

Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector

Three female patients with osteoarthrotic hips received total hip replacement arthroplasties after failed rotational acetabular osteotomies (RAO) were reported. In the first case, there was necrosis of the thin acetabular fragment and a collapse of the large grafted iliac bone because of technical problems. The second case had residual development dislocation of the hip preoperatively which resulted in pseudoarthrosis and instability of the pubic bone postoperatively. This patient was considered to be a bad candidate for rotational acetabular osteotomy. The last case was 65 years old, too old to treat by osteotomy. Deterioration of the articular cartilage was expected. All of them were successfully treated with total hip arthroplasties. The ages of the patients, the stage of osteoarthrosis, the thickness of the osteotomized acetabular fragment, and the size of the grafted bone seemed to be factors influencing the outcome of the RAO.     

9-azidoacridine, a new photoaffinity label for nucleotide- and aromatic-binding sites in proteins

The effect of the photolytic reagent 9-azidoacridine, optionally 3H-labelled, was studied both kinetically and structurally on nine different enzymes, namely alpha-chymotrypsin, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, alanine dehydrogenase, D-amino acid oxidase, ribonuclease A, alkaline phosphatase and alpha-amylase. Dark inhibition was observed in several cases. The concentration of the inhibitor ranged from 0.2 microM to 0.67 microM and demonstrated competitive kinetics with nucleotide cofactors when present. All concentrations of inhibitor showed increased inhibition on photolysis. Examination of the oligopeptides from hydrolysis of the covalently 3H-labelled derivative in conjunction with known amino acid sequence and tertiary structure established that the primary site of interaction in those cases for which the tertiary structure was available involved the active-site region. The above results in conjunction with those obtained with the structural analogues 9-aminoacridine and 9-amino-1,2,3,4-tetrahydroacridine established that this reagent acts as a molecular probe of aromatic- and, in particular, nucleotide-binding sites. This reagent provides a further additional method for studying the nucleotide cofactor domain.     

Gbp1p, a protein with RNA recognition motifs, binds single-stranded telomeric DNA and changes its binding specificity upon dimerization

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.     

Kinase suppressor of Ras inhibits the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase by growth factors, activated Ras, and Ras effectors

Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. We examined the effect of mammalian KSR expression on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibited the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrated that the KSR kinase domain was necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibited cell transformation by activated RasVal-12 but had no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.     

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.     

Probing the structure and mechanism of Ras protein with an expanded genetic code

Mutations in Ras protein at positions Gly12 and Gly13 (phosphate-binding loop L1) and at positions Ala59, Gly60, and Gln61 (loop L4) are commonly associated with oncogenic activation. The structural and catalytic roles of these residues were probed with a series of unnatural amino acids that have unusual main chain conformations, hydrogen bonding abilities, and steric features. The properties of wild-type and transforming Ras proteins previously thought to be uniquely associated with the structure of a single amino acid at these positions were retained by mutants that contained a variety of unnatural amino acids. This expanded set of functional mutants provides new insight into the role of loop L4 residues in switch function and suggests that loop L1 may participate in the activation of Ras protein by effector molecules.     

Mammalian cytidine 5'-monophosphate N-acetylneuraminic acid synthetase: a nuclear protein with evolutionarily conserved structural motifs

Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is species- and tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMP-Neu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the lec32 mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.     

Gem: an induced, immediate early protein belonging to the Ras family

A gene encoding a 35-kilodalton guanosine triphosphate (GTP)-binding protein, Gem, was cloned from mitogen-induced human peripheral blood T cells. Gem and Rad, the product of a gene overexpressed in skeletal muscle in individuals with Type II diabetes, constitute a new family of Ras-related GTP-binding proteins. The distinct structural features of this family include the G3 GTP-binding motif, extensive amino- and carboxyl-terminal extensions beyond the Ras-related domain, and a motif that determines membrane association. Gem was transiently expressed in human peripheral blood T cells in response to mitogenic stimulation; the protein was phosphorylated on tyrosine residues and localized to the cytosolic face of the plasma membrane. Deregulated Gem expression prevented proliferation of normal and transformed 3T3 cells. These results suggest that Gem is a regulatory protein, possibly participating in receptor-mediated signal transduction at the plasma membrane.     

A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice

Three radiation-induced alleles of the mouse p locus, p6H, p25H, and pbs, cause defects in growth, coordination, fertility, and maternal behavior in addition to p gene-related hypopigmentation. These alleles are associated with disruption of the p gene plus an adjacent gene involved in the disorders listed. We have identified this adjacent gene, previously named rjs (runty jerky sterile), by positional cloning. The rjs cDNA is very large, covering 15,264 nucleotides. The predicted rjs-encoded protein (4,836 amino acids) contains several sequence motifs, including three RCC1 repeats, a structural motif in common with cytochrome b5, and a HECT domain in common with E6-AP ubiquitin ligase. On the basis of sequence homology and conserved synteny, the rjs gene is the single mouse homolog of a previously described five- or six-member human gene family. This family is represented by at least two genes, HSC7541 and KIAA0393, from human chromosome 15q11-q13. HSC7541 and KIAA0393 lie close to, or within, a region commonly deleted in most Prader-Willi syndrome patients. Previous work has suggested that the multiple phenotypes in rjs mice might be due to a common neuroendocrine defect. In addition to this proposed mode of action, alternative functions of the rjs gene are evaluated in light of its known protein homologies.     

Primary structure of Drosophila ribosomal protein L14 and identification of conserved protein motifs

Determination of the primary structure of individual ribosomal proteins is important for understanding their functions and organization within the ribosome. I have sequenced a cDNA that encodes a Drosophila homolog of the rat ribosomal protein L14. The cDNA sequence was 601 nucleotides long, with an open reading frame encoding a protein of 166 amino acids. Homology searches revealed 34-38% sequence identity to the rat and yeast L14 ribosomal proteins. There were also extensive homologies to sequences in the EST database, which are likely to encode portions of L14. Analysis of sequence comparisons revealed several highly conserved regions, one of which is related to a portion of ribosomal protein L27. The sizes of the L14 proteins vary between different species, with most of the variability confined to the C-terminal region.     

Investigation of a rifampin, fusidic-acid and mupirocin releasing silicone catheter

After strict hygienical measures have been exhausted the use of plastic materials with antibacterial activity may reduce catheter related-bacterial colonization. An antimicrobial silicone catheter was investigated by HPLC-measurement, SEM, antimicrobial assays and standard biocompatibility tests. The modified catheter was highly biocompatible and the antimicrobial leaching non-toxic. The initially release rate was governed by the drug solubility in the 'sink' and surface loading ('burst effect'). The second continuous period depended on the drug velocity in the silicone matrix and was extended up to 100 days with a proportionality to square root of t for each drug. Diffusion exponents were in range of 2 x 10(-8) to 1 x 10(-9) (cm2 sec(-1)). The lower diffusion exponent of mupirocin was explained by its higher cohesion energy and lower physico-chemical compatibility with the embedding silicone. The antimicrobial drugs were in a molecular-dispersed state with the silicone-matrix, whereas superficially located crystals of the antibiotics covering the catheter surface could be demonstrated by SEM.     

Analysis of polymorphisms of the 2'',3''-cyclic nucleotide-3''-phosphodiesterase gene in patients with multiple sclerosis

Susceptibility to multiple sclerosis (MS) is widely held to have a genetic component. Possible candidate genes conferring this susceptibility include those coding for proteins specific to central nervous system (CNS) myelin. 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) is an enzyme found at high concentrations in CNS myelin, however its function is unknown. The amino acid sequence of CNPase shows a C-terminal motif characteristic of proteins involved in signal transduction pathways, suggesting a key role in myelin function. We have analysed the entire expressed sequence of the human CNPase gene in patients with multiple sclerosis and in healthy controls using single strand conformation polymorphism (SSCP) analysis. Nine previously undescribed mutations were detected, most of these occurred with equal frequency in both groups. However, a T-->C transition at nucleotide position 4306 in the region of the gene coding for the 3' untranslated region of the mature mRNA was found in a homozygous form in two out of 54 patients but in none of 100 controls. While the significance of this is unclear, it would appear unlikely that mutations in the expressed regions of the human CPNase gene contribute to genetic susceptibility to MS in the majority of sufferers.     

Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1-120) and oligomerization (amino acids 134-181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitro experiments demonstrated that a pair of predicted alpha-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions.     

GCKR links the Bcr-Abl oncogene and Ras to the stress-activated protein kinase pathway

The Bcr-Abl oncogene, found in Philadelphia chromosome-positive myelogenous leukemia (CML), activates Ras and triggers the stress-activated protein kinase (SAPK or Jun NH2-terminal kinase [JNK]) pathway. Interruption of Ras or SAPK activation dramatically reduces Bcr-Abl-mediated transformation. Here, we report that Bcr-Abl through a Ras-dependent pathway signals the serine/threonine protein kinase GCKR (Germinal Center Kinase Related) leading to SAPK activation. Either an oncogenic form of Ras or Bcr-Abl enhances GCKR catalytic activity and its activation of SAPK, whereas inhibition of GCKR impairs Bcr-Abl-induced SAPK activation. Bcr-Abl mutants that are impaired for GCKR activation are also unable to activate SAPK. Consistent with GCKR being a functional target in CML, GCKR is constitutively active in CML cell lines and found in association with Bcr-Abl. Our results indicate that GCKR is a downstream target of Bcr-Abl and strongly implicate GCKR as a mediator of Bcr-Abl in its transformation of cells.     

The alternative H-ras protein p19 displays properties of a negative regulator of p21Ras

In previous work, we demonstrated that expression of mammalian H-ras genes is controlled by alternative splicing. Mutational analyses indicated that most H-ras premessenger RNA (pre-mRNA) is not processed into mRNA for the usual p21Ras protein, but is recognized instead for processing through an alternative pathway that would produce mRNA for a hypothetical p19Ras protein, which would be a truncated form of p21 with a different carboxyl-terminus. We have raised the possibility that p19 could be a negative regulator of p21, but only small amounts of mature p19 mRNA could be detected and we had no evidence that the protein could be made. We now show by transient and stable transfection experiments that p19 can be produced from complementary DNA (cDNA) expression vectors in human 293 cells, which express early adenoviral genes. However, p19 cDNA inhibited the formation of drug-selected colonies of Rat-1 cells and surviving colonies produced little p19. Colony formation and p19 expression were increased when the cDNA had mutations in the putative effector domain of p19 or when oncogenes believed to act downstream of p21Ras, such as adenoviral E1A, were included in the transfections. These results indicate that p19 can act as an inhibitor of p21. We also show that authentic p19 can be abundantly expressed in 293 cells from H-ras gene constructs with mutations favoring the alternative splicing pathway, which suggests that regulated splicing could support overproduction of p19 under natural conditions. Our work indicates that mutations abolishing alternative H-ras splicing may not only contribute to oncogenesis by increasing the production of p21, but also by interfering with the production of an antiproliferative H-ras activity.