Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system

The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed.     

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.     

Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18-->e mutation in leukemic cells

Op18/stathmin (Op18) is a phosphorylation-regulated microtubule destabilizer that is frequently overexpressed in tumors. The importance of Op18 in malignancy was recently suggested by identification of a somatic Q18-->E mutation of Op18 in an adenocarcinoma. We addressed the functional consequences of aberrant Op18 expression in leukemias by analyzing the cell cycle of K562 cells either depleted of Op18 by expression of interfering hairpin RNA or induced to express wild-type or Q18E substituted Op18. We show here that although Op18 depletion increases microtubule density during interphase, the density of mitotic spindles is essentially unaltered and cells divide normally. This is consistent with phosphorylation-inactivation of Op18 during mitosis. Overexpression of wild-type Op18 results in aneugenic activities, manifest as aberrant mitosis, polyploidization, and chromosome loss. One particularly significant finding was that the aneugenic activity of Op18 was dramatically increased by the Q18-->E mutation. The hyperactivity of mutant Op18 is apparent in its unphosphorylated state, and this mutation also suppresses phosphorylation-inactivation of the microtubule-destabilizing activity of Op18 without any apparent effect on its phosphorylation status. Thus, although Op18 is dispensable for mitosis, the hyperactive Q18-->E mutant, or overexpressed wild-type Op18, exerts aneugenic effects that are likely to contribute to chromosomal instability in tumors.     

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.     

Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells

The microtubule cytoskeleton is differentially regulated by a diverse array of proteins during interphase and mitosis. Op18/stathmin (Op18) and microtubule-associated protein (MAP)4 have been ascribed opposite general microtubule-directed activities, namely, microtubule destabilization and stabilization, respectively, both of which can be inhibited by phosphorylation. Here, using three human cell models, we depleted cells of Op18 and/or MAP4 by expression of interfering hairpin RNAs and we analyzed the resulting phenotypes. We found that the endogenous levels of Op18 and MAP4 have opposite and counteractive activities that largely govern the partitioning of tubulin dimers in the microtubule array at interphase. Op18 and MAP4 were also found to be the downstream targets of Ca(2+)- and calmodulin-dependent protein kinase IV and PAR-1/MARK2 kinase, respectively, that control the demonstrated counteractive phosphorylation-mediated regulation of tubulin dimer partitioning. Furthermore, to address mechanisms regulating microtubule polymerization in response to cell signals, we developed a system for inducible gene product replacement. This approach revealed that site-specific phosphorylation of Op18 is both necessary and sufficient for polymerization of microtubules in response to the multifaceted signaling event of stimulation of the T cell antigen receptor complex, which activates several signal transduction pathways.     

Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells

Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.     

Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3).

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1alpha,25-dihydroxyvitamin D(3). We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50-55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.     

Avian acute leukemia virus OK10 has an 8.2-kilobase genome and modified glycoprotein gp 78

We have analyzed the structure of OK10-BM virus, an avian acute leukemia virus produced by a bone marrow-derived cell line of macrophage origin, and compared it with that of OK10 AV, an associated virus originally present in the OK10 virus stock. The RNAs of OK10-BM virus and OK10 AV had the same mobility in agarose gels, corresponding to 8.0 to 8.5 kilobases, a size considerably larger than that of the transforming component (5 to 6 kb) of most other avian acute leukemia viruses. Fingerprint analysis showed a close relationship between OK10-BM virus and OK10 AV RNAs. The polypeptide compositions of OK10-BM and OK10 AV viruses were similar except for the envelope glycoproteins. In analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the large envelope glycoprotein of OK10-BM virus migrated at M_r = 78,000 (gp78), whereas OK10 AV had the characteristic 85,000-dalton glycoprotein (gp85) of nondefective avian leukemia viruses. gp78 was weakly labeled with methionine, glycine, proline, or mannose, suggesting that purified OK10-BM virus had reduced amounts of the modified envelope glycoprotein. In cell-free rabbit reticulocyte lysates, OK10-BM virion RNA directed the synthesis of a 200,000-dalton polypeptide (p200), a 180,000-dalton polypeptide (pr180), and a 76,000-dalton polypeptide (pr76), whereas OK10 AV RNA gave rise only to pr180 and pr76, suggesting that p200 may represent an OK10-BM-encoded transforming protein. No biochemical evidence for the presence of an associated helper virus was found in the OK10-BM virus population produced by the macrophage cell line. However, when OK10-BM virus was serially passaged in chicken embryo fibroblasts, a virus having structural properties similar to those of OK10 AV (OK10 AV-specific oligonucleotides and gp85) appeared after three passages. Moreover, nonproducer clones of transformed cells could be readily obtained in OK10-BM virus-infected quail cell cultures. It is thus likely that the bone marrow-derived macrophage cell line produces a transforming virus defective in its env gene and low amounts of an associated helper virus, which upon transfer to fibroblasts is preferentially replicated.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Molecular cloning of subunits of complex I from Neurospora crassa. Primary structure and in vitro expression of a 22-kDa polypeptide

A lambda gt11 cDNA expression library was screened with antibodies directed against individual subunits of complex I from Neurospora crassa. Clones encoding cytoplasmically synthesized polypeptides with apparent molecular masses of 22, 29, 31, and 33 kDa were isolated. Northern blot analysis revealed that the corresponding genes are transcribed into mRNA species of about 0.85, 0.95, 1.3, and 1.4 kilobases, respectively. Further characterization of clones encoding the 22-kDa subunit was performed. A cDNA insert of 755 base pairs containing the complete coding sequence was used to express the polypeptide in vitro. A precursor of the protein is synthesized on cytoplasmic ribosomes without a cleavable signal sequence. Our data indicate that after import into the organelle and before assembly into complex I, the 22-kDa polypeptide forms intramolecular disulfide bridge(s). Nucleotide sequencing revealed an open reading frame coding for a protein of 183 amino acids. A molecular mass of 20,828 daltons was calculated. The polypeptide is hydrophilic and contains no obvious membrane-spanning domains. Eight cysteine residues arranged in a regular pattern are found in the primary structure of the protein. Therefore, this subunit is a good candidate to bind at least one of the iron-sulfur centers present in complex I of the respiratory chain.     

Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene

We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors

The myeloproliferative leukemia virus (MPLV) is an acute leukemogenic murine replication-defective retrovirus. By sequencing the envelope gene of a biologically active MPLV clone, we found that this region comprises a novel oncogene named v-mpl in phase with two parts of the Friend murine leukemia virus envelope gene. The MPLV env region could encode an env-mpl fusion polypeptide that presents the characteristics of a transmembrane protein. We show that in vitro infection of bone marrow cells with helper-free MPLV readily yields immortalized factor-independent hematopoietic cell lines of different lineages. In mice, the c-mpl proto-oncogene is expressed in hematopoietic tissues as a 3 kb mRNA. Since v-mpl shares strong structural analogies with the hematopoietin receptor superfamily, it is likely that MPLV has transduced a truncated form of an as yet unidentified hematopoietic growth factor receptor.     

Isolation of a cDNA clone for human antithrombin III

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.     

Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.     

Stathmin/Op18 phosphorylation is regulated by microtubule assembly

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of "mitotic chromatin." Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.