The Cdc6 Nucleotide–Binding Site Regulates Its Activity in DNA Replication in Human Cells

The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.     

Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase , which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.     

A serine/threonine kinase which causes apoptosis-like cell death interacts with a calcineurin B-like protein capable of binding Na(+)/H(+) exchanger

We surveyed proteins capable of binding to the cytoplasmic domain of Na(+)/H(+) exchanger (NHE)1 in a rat brain cDNA library with the yeast two-hybrid system. One clone obtained coded for a protein reported previously as a human calcineurin homologous protein (CHP). Since CHP is homologous to the regulatory subunit B of calcineurin, we expected a possible interacting partner of CHP like the catalytic subunit of calcineurin (calcineurin A), and surveyed this putative partner again with the yeast two-hybrid system. A clone thus obtained coded for a kinase, which is basically the same as that reported for human DRAK2. Overexpression of the rat homologue of DRAK2 caused apoptosis-like cell death of NIH3T3 cells, which was dependent on the kinase activity, confirming the previous result for DRAK2. The purified CHP and rat DRAK2 proteins synthesized in Escherichia coli could bind in vitro. CHP and rat DRAK2 expressed in COS-7 cells were found to be localized in the Golgi apparatus and nucleus, respectively. Some of them was also found in the membrane peripheral region. When they were co-expressed in the same cells, most of CHP moved to the nucleus where rat DRAK2 is located, suggesting in vivo interaction of these proteins. However, minor but significant fractions of both proteins were also found in the membrane peripheral region. Rat DRAK2 is expressed highly in thymus, spleen, and testis, where the apoptosis plays an important role in physiology.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.     

Studies on Rabies Virus RNA Polymerase: 1. cDNA Cloning of the Catalytic Subunit (L Protein) of Avirulent HEP-Flury Strain and Its Expression in Animal Cells

To investigate the RNA polymerase of rabies virus, we cloned a cDNA of the catalytic subunit (called L protein because of its large molecular size) of the HEP-Flury strain, an avirulent strain obtained by high frequencies of serial embryonated hen egg passages. Nucleotide sequencing showed that the cDNA encodes a long polypeptide of 2,127 amino acids (Mr. 242,938). A comparison of the deduced amino acid sequence with that of other strains (PV and SAD B19) indicated that the sequence was highly conserved, except for several amino acid substitutions which were accumulated in some limited regions. A fragment of the cDNA was used for expression in Escherichia coli (E. coli) to prepare the L antigen for raising the antibodies in rabbits. Immunoprecipitation studies with the rabbit antiserum showed that the polypeptides produced in the L cDNA-transfected COS-7 cells displayed almost the same electrophoretic mobility as that of authentic L protein. Immunofluorescence studies indicated that both L and P (another subunit of RNA polymerase) proteins displayed colocalized distribution with the nucleocapsid antigen (N) in the cytoplasmic inclusion bodies, where envelope proteins (G and M) were absent. On the other hand, expression of the L protein alone did not cause inclusion body-like granular distribution, suggesting that the inclusion body-like accumulation depends on certain interaction(s) with other viral gene products, probably with the ribonucleoproteins comprising the inclusion bodies.     

Cloning and characterization of a cDNA encoding bovine mannan-binding protein

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.     

PR48, a novel regulatory subunit of protein phosphatase 2A, interacts with Cdc6 and modulates DNA replication in human cells

Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified. A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait. PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A. PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G(1) arrest. These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.     

Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase alpha.

Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7.     

Characterisation of XlCdc1, a Xenopus homologue of the small (PolD2) subunit of DNA polymerase delta; identification of ten conserved regions I-X based on protein sequence comparisons across ten eukaryotic species

DNA polymerase delta (Pol delta), which plays keys roles in DNA replication, repair and recombination in eukaryotic cells, comprises at least two essential subunits - a large catalytic subunit (PolD1) possessing both DNA polymerase and 3'-5' exonuclease activities, and a smaller subunit (PolD2) whose function is not yet clear. Here we describe the cloning and sequencing of a Xenopus cDNA encoding a homologue of the PolD2 subunit. This protein (designated XlCdc1) is 69% identical to the human PolD2 protein and 34% identical to fission yeast Cdc1. Alignment of PolD2 protein sequences across ten eukaryotic species identifies 36 invariant amino-acid positions. These 36 residues are located within ten conserved regions (designated I-X) likely to have key functional roles. Consistent with this, the mutations in six previously identified yeast mutant PolD2 proteins map within conserved regions III, VI, VII and VIII. Several of the invariant amino acids are also conserved across the archaeal DNA polymerase II DP1 protein family.     

Cloning and characterization of a mammalian prenyl protein-specific protease

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.