Characterization of the KIF3C Neural Kinesin-like Motor from Mouse

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of axonal transport, a cDNA encoding a new kinesin-like protein called KIF3C was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KIF3C is a member of the KIF3 family. In contrast to KIF3A and KIF3B, Northern and Western analysis indicated that KIF3C expression is highly enriched in neural tissues such as brain, spinal cord, and retina. When anti-KIF3C antibodies were used to stain the cerebellum, the strongest signal came from the cell bodies and dendrites of Purkinje cells. In retina, anti-KIF3C mainly stains the ganglion cells. Immunolocalization showed that the KIF3C motor in spinal cord and sciatic nerve is mainly localized in cytoplasm. In spinal cord, the KIF3C staining was punctate; double labeling with anti-giantin and anti-KIF3C showed a clear concentration of the motor protein in the Golgi complex. Staining of ligated sciatic nerves demonstrated that the KIF3C motor accumulated at the proximal side of the ligated nerve, which suggests that KIF3C is an anterograde motor. Immunoprecipitation experiments revealed that KIF3C and KIF3A, but not KIF3B, were coprecipitated. These data, combined with previous data from other labs, indicate that KIF3C and KIF3B are “variable” subunits that associate with a common KIF3A subunit, but not with each other. Together these results suggest that KIF3 family members combinatorially associate to power anterograde axonal transport.

Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian CellsV⃞

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Identification of Darlin, a Dictyostelium Protein with Armadillo-like Repeats that Binds to Small GTPases and Is Important for the Proper Aggregation of Developing Cells

We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development.

Noninvasive measurement of gene expression in skeletal muscle

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using 31P-magnetic resonance spectroscopy (31P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique 31P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 ± 0.90 and 13.6 ± 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated in vivo by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 ± 0.06, PCr/ATP was decreased by 77 ± 8%, whereas PArg/ATP was decreased by 50 ± 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

A small nucleolar RNA requirement for site-specific ribose methylation of rRNA in Xenopus

Vertebrate cells contain a large number of small nucleolar RNA (snoRNA) species, the vast majority of which bind fibrillarin. Most of the fibrillarin-associated snoRNAs can form 10- to 21-nt duplexes with rRNA and are thought to guide 2′-O-methylation of selected nucleotides in rRNA. These include mammalian UHG (U22 host gene)-encoded U25–U31 snoRNAs. We have characterized two novel human snoRNA species, U62 and U63, which similarly exhibit 15- (with one interruption) and 12-nt complementarities and are therefore predicted to direct 2′-O-methylation of A590 in 18S and A4531 in 28S rRNA, respectively. To establish the function of antisense snoRNAs in vertebrates, we exploited the Xenopus oocyte system. Cloning of the Xenopus U25–U31 snoRNA genes indicated that they are encoded within multiple homologs of mammalian UHG. Depletion of U25 from the Xenopus oocyte abolished 2′-O-methylation of G1448 in 18S rRNA; methylation could be restored by injecting either the Xenopus or human U25 transcript into U25-depleted oocytes. Comparison of Xenopus and human U25 sequences revealed that only boxes C, D, and D′, as well as the 18S rRNA complement, were invariant, suggesting that they may be the only elements required for U25 snoRNA stability and function.

A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins

The Krüppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys2-His2 type. Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region. KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys3HisCys4, family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction. The KRIP-1 protein has high homology to TIF1, a putative modulator of ligand-dependent activation function of nuclear receptors. A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied. When a GAL4–KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription. Thus, KRIP-1 interacts with the KRAB-A region of C2H2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185neu. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185neu-treated tumor cells but not in cells expressing internalization defective p185neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement

As an adhesion receptor, the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5,6-dichloro-1-β-d-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3′-untranslated region of the uPAR cDNA into a serum-inducible rabbit β-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3′-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3′ AU-rich elements.

Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2

The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A2 (sPLA2), this gene was referred to as PLA2-like (PLA2L) and enabled the identification of human Oc90. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA2. The cloning of Oc90 will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1

We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376–405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380–408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein–protein interactions may not be necessary for this interaction of chHAT-1.

Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.