CD39L2, a gene encoding a human nucleoside diphosphatase, predominantly expressed in the heart

E-NTPDases are extracellular enzymes that hydrolyze nucleotides. The human E-NTPDase gene family currently consists of five reported members (CD39, CD39L1, CD39L2, CD39L3, and CD39L4). Both membrane-bound and secreted family members have been predicted by encoded transmembrane and leader peptide motifs. In this report, we demonstrate that the human CD39L2 gene is expressed predominantly in the heart. In situ hybridization results from heart indicate that the CD39L2 message is expressed in muscle and capillary endothelial cells. We also show that the CD39L2 gene encodes an extracellular E-NTPDase. Flow cytometric experiments show that transiently expressed CD39L2 is present on the surface of COS-7 cells. Transfected cells also produce recombinant glycosylated protein in the medium, and this process can be blocked by brefeldin A, an inhibitor of the mammalian secretory pathway. The enzymology of CD39L2 shows characteristic features of a typical E-NTPDase, but with a much higher degree of specificity for NDPs over NTPs as enzymatic substrates. The kinetics of the ADPase activity exhibit positive cooperativity. The predominance of CD39L2 expression in the heart supports a functional role in regulating platelet activation and recruitment in this organ.     

Secretion of human glucocerebrosidase from stable transformed insect cells using native signal sequences.

The lysosomal hydrolase, glucocerebrosidase (GBA), catalyses the penultimate step in the breakdown of membrane glycosphingolipids. An inherited deficiency of this enzyme activity leads to the onset of Gaucher disease, the most common lysosomal storage disorder. Affected individuals range from adults with hepatosplenomegaly, haematological complications, and bone pain (type 1 disease) to children and neonates with severe neuronopathy leading to neurological degradation and premature death (type 2 and type 3 disease). Enzyme replacement therapy has become the standard of treatment for type I Gaucher disease but remains an expensive option, in part because of the cost of recombinant enzyme production using mammalian cell culture. Using a nonlytic integrative plasmid expression system, we have successfully produced active human GBA in stable transformed Sf9 (Spodoptera frugiperda) cells. Both the 39 and 19 amino acid native GBA signal sequences were capable of endoplasmic reticulum targeting, which led to secretion of the recombinant protein, although approximately 30% more enzyme was produced using the longer signal sequence. The secreted product was purified to apparent electrophoretic homogeneity using hydrophobic interaction chromatography and found to be produced in a fully glycosylated and a hypoglycosylated form, both of which cross-reacted with a human GBA-specific monoclonal antibody. The pH optimum (at pH 5.5) for activity of the recombinant enzyme was as expected for human GBA using the artificial substrate 4-methyl-umbelliferyl-beta-D-glycopyranoside. With initial nonoptimized expression levels estimated at 10-15 mg/L using small-scale batch cultures, stable transformed insect cells could provide a viable alternative system for the heterologous production of human GBA when grown under optimized perfusion culture conditions.     

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and CD4 binding

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

Role of oligosaccharides in the immune response of sheep vaccinated with Lucilia cuprina larval glycoprotein, peritrophin-95

The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep. The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen. This protein induced an immune response in vaccinated sheep that inhibited larval growth. Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells. The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded. Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity. Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement. The activity was associated with IgG(1) and IgG(2) but not IgM. There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein. Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies. Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95. It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.     

CD39-L4 is a secreted human apyrase, specific for the hydrolysis of nucleoside diphosphates.

The human ecto-apyrase gene family consists of five reported members (CD39, CD39-L1, CD39-L2, CD39-L3, and CD39-L4). The family can be subdivided into two groups by conservation of proposed structural domains. The CD39, CD39-L1, and CD39-L3 genes all encode hydrophobic portions in their carboxy and amino termini, serving as transmembrane domains for CD39 and potentially for the other two members. CD39-L2 and CD39-L4 genes encode hydrophobic portions in their amino termini, suggesting that they might encode secreted apyrases. We demonstrate that the CD39-L4 gene encodes the first reported human secreted ecto-apyrase. COS-7 cells transfected with a CD39-L4 expression construct utilizing the naturally occurring leader peptide express recombinant protein outside of the cells. This expression can be blocked by brefeldin A, a chemical that inhibits a step in mammalian secretory pathways. We also demonstrate expression of CD39-L4 message in macrophages, suggesting that the protein is present in the circulation. Furthermore, we show that CD39-L4 is an E-type apyrase, is dependent on calcium and magnesium cations, and has high degree of specificity for NDPs over NTPs as enzymatic substrates. A potential physiological role in hemostasis and platelet aggregation is presented.     

Functionality of insect‐cell‐derived colorectal cancer vaccine candidate protein EpCAM‐Fc in human dendritic cells

The baculovirus–insect cell expression system is widely used to produce recombinant proteins for various biomedical applications. Our previous study demonstrated that EpCAM, a colorectal cancer vaccine candidate protein, can be expressed in the baculovirus–insect cell expression system. However, its functionality (the ability to elicit an immune response), which is important for its possible use as a colorectal cancer vaccine for immunotherapy, still needed to be confirmed. In this study, we examined the ability of recombinant EpCAM to induce maturation of immature dendritic cells (DCs) derived from CD34⁺ cells isolated from human umbilical cord blood. We demonstrated that EpCAM induces the expression of four DC maturation markers: CD80, CD83, CD86 and MHC II. These results suggest that EpCAM produced in the baculovirus–insect cell expression system is functional in terms of its ability to trigger maturation of human DCs.     

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.     

Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein.     

Characterization of the oligosaccharide structures associated with the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane-associated glycoprotein. The protein can exist in three different molecular weight forms of approximately 127, 131, and 160 kDa, representing either nonglycosylated, core glycosylated, or fully mature, complex glycosylated CFTR, respectively. The most common mutation in cystic fibrosis (CF) results in the synthesis of a variant (DeltaF508-CFTR) that is incompletely glycosylated and defective in its trafficking to the cell surface. In this study, we have analyzed the oligosaccharide structures associated with the different forms of recombinant CFTR, by expressing and purifying the channel protein from either mammalian Chinese hamster ovary (CHO) or insect Sf9 cells. Using glycosidases and FACE analysis (fluorophore-assisted carbohydrate electrophoresis) we determined that purified CHO-CFTR contained polylactosaminoglycan (PL) sequences, while Sf9-CFTR had only oligomannosidic saccharides with fucosylation on the innermost GlcNAc. The presence of PL sequences on the recombinant CHO-CFTR is consistent with a normal feature of mammalian processing, since endogenous CFTR isolated from T84 cells displayed a similar pattern of glycosylation. The present study also reports on the use of FACE for the qualitative analysis of small amounts of glycoprotein oligosaccharides released enzymatically.     

鸡源CD40L基因克隆、蛋白表达及生物活性检测

为获得鸡源CD40L (chCD40L) 蛋白,以鸡脾细胞制备cDNA并以之为模板扩增chCD40L基因,构建pFastBac-chCD40L供体重组质粒,转化感受态细胞DH10Bac,通过筛选及鉴定获得Bacmid-chCD40L重组质粒,转入真核表达系统sf9昆虫细胞进行蛋白表达与纯化,获得His-chCD40L蛋白。此外,构建pQM01-chCD40L质粒,转染HEK 293T细胞进行蛋白表达与纯化,获得Strep-chCD40L蛋白。亲和层析纯化的chCD40L蛋白浓度为0.01 mg/mL。为检测chCD40L蛋白的生物活性,分离和培养3周龄SPF雏鸡的法氏囊组织原代细胞,将chCD40L加入细胞培养液刺激细胞增殖,通过Western blotting试验、间接免疫荧光试验、流式细胞术检测,发现该蛋白能够与法氏囊B淋巴细胞表面的CD40结合,说明chCD40L具有生物活性。成功获得chCD40L蛋白,为原代B淋巴细胞体外培养及IBDV野毒分离与诊断奠定了基础。     

Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity.

Platelet glycoprotein (GP) Ibalpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of approximately 300 residues of GPIbalpha mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbalpha, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibalpha to these ligands.     

The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.

DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.     

Expression and Purification of Chaperone-Active Recombinant Clusterin

Clusterin was the first described secreted mammalian chaperone and is implicated as being a key player in both intra- and extracellular proteostasis. Its unique combination of structural features and biological chaperone activity has, however, previously made it very challenging to express and purify the protein in a correctly processed and chaperone-active form. While there are multiple reports in the literature describing the use of recombinant clusterin, all of these reports suffer from one or more of the following shortcomings: details of the methods used to produce the protein are poorly described, the product is incompletely (if at all) characterised, and purity (if shown) is in many cases inadequate. The current report provides the first well validated method to economically produce pure chaperone-active recombinant clusterin. The method was developed after trialling expression in cultured bacterial, yeast, insect and mammalian cells, and involves the expression of recombinant clusterin from stably transfected HEK293 cells in protein-free medium. The product is expressed at between 7.5 and 10 µg/ml of culture, and is readily purified by a combination of immunoaffinity, cation exchange and size exclusion chromatography. The purified product was shown to be glycosylated, correctly proteolytically cleaved into α- and β-subunits, and have chaperone activity similar to that of human plasma clusterin. This new method creates the opportunity to use mutagenesis and metabolic labelling approaches in future studies to delineate functionally important sites within clusterin, and also provides a theoretically unlimited supply of recombinant clusterin which may in the future find applications in the development of therapeutics.     

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.     

Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.

The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. Previously, we identified a human Cdc7 homolog, HsCdc7. In this study, we report the identification of a human Dbf4 homolog, HsDbf4. We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells. HsDbf4 protein levels are regulated during the cell cycle with a pattern that matches that of HsCdc7 protein kinase activity. They are low in G(1), increase during G(1)-S, and remain high during S and G(2)-M. Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro. Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo (32)P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MCM2 is a physiological HsCdc7-HsDbf4 substrate. Immunoneutralization of HsCdc7-HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication. These results indicate that the HsCdc7-HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells.     

Identification and characterization of uncoupling protein 4 in fat body and muscle mitochondria from the cockroach <Emphasis Type="Italic">Gromphadorhina cocquereliana</Emphasis>

We have identified and characterized an uncoupling protein in mitochondria isolated from leg muscle and from fat body, an insect analogue tissue of mammalian liver and adipose tissue, of the cockroach Gromphadorhina coquereliana (GcUCP). This is the first functional characterization of UCP activity in isolated insect mitochondria. Bioenergetic studies clearly indicate UCP function in both insect tissues. In resting (non-phosphorylating) mitochondria, cockroach GcUCP activity was stimulated by the addition of micromolar concentrations of palmitic acid and inhibited by the purine nucleotide GTP. Moreover, in phosphorylating mitochondria, GcUCP activity was able to divert energy from oxidative phosphorylation. Functional studies indicate a higher activity of GcUCP-mediated uncoupling in cockroach muscle mitochondria compared to fat body mitochondria. GcUCP activation by palmitic acid resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of UCPs in insects. GcUCP protein was immunodetected using antibodies raised against human UCP4 as a single band of around 36 kDa. GcUCP protein expression in cockroach muscle mitochondria was significantly higher compared to mitochondria isolated from fat body. LC-MS/MS analyses revealed 100% sequence identities for peptides obtained from GcUCP to UCP4 isoforms from D. melanogaster (the highest homology), human, rat or other insect mitochondria. Therefore, it can be proposed that cockroach GcUCP corresponds to the UCP4 isoforms of other animals.     

Purification and characteristics of recombinant human folylpoly-gamma-glutamate synthetase expressed at high levels in insect cells

Folylpoly-gamma-glutamate synthetase activity is central to the operation of folate metabolism and is essential for the survival of mammalian stem cell populations but the very low levels of endogenous expression of this enzyme have greatly limited its study. We now report the expression of cytosolic folylpoly-gamma-glutamate synthetase (FPGS) cloned from human leukemic cells in baculovirus-infected insect cells at levels of 4-5% of the total soluble protein of the cells. As was the case with endogenously expressed mammalian FPGS, recombinant enzyme was quantitatively blocked at the amino terminus in spite of the large-scale production in insect cells. A three-step purification procedure resulted in an overall yield of 7-35 mg per liter of culture with a recovery of about 50% and purity approximately 95%; pure enzyme was stable to storage for extended periods. Pure protein had a specific activity of 25 micromol h(-1)mg(-1) with aminopterin as a substrate and used a broad spectrum of folates as substrates. The pure enzyme also carried out ATP hydrolysis in the absence of a folate substrate or glutamic acid; this partial reaction occurred at a k(cat) about 0.4% that of the full reaction. In vitro, this single protein added several (1-8) moles of glutamic acid per mole of folate analog, the same spectrum of folate polyglutamates as seen in vivo. The quantities of pure enzyme achievable in insect cells should allow functional and structural studies on this enzyme.     

Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue.

The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain.