scholarly journals A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635 ◽  
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

scholarly journals Functional Domains of Fused, a Serine-Threonine Kinase Required for Signaling in Drosophila

Genetics ◽  
1996 ◽  
Vol 142 (4) ◽  
pp. 1181-1198
Author(s):  
Pascal Thérond ◽  
Georges Alves ◽  
Bernadette Limbourg-Bouchon ◽  
Hervé Tricoire ◽  
Elizabeth Guillemet ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Kinase Domain ◽  
In Vitro Mutagenesis ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Terminal Domain ◽  
Segment Polarity ◽  
Protein Functions

Abstract fused (fu) is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles (fu0, fuI, fuII). Here, we report the molecular analysis of known fu mutations and the generation of new alleles by in vitro mutagenesis. We show that the Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu+ activity while a central region appears to be dispensable. We observe a striking correlation between the molecular lesions of fu mutations and the phenotype displayed in their interaction with Su(fu). Indeed, fuI alleles which are suppressed by Su(fu) mutations are defined by inframe alterations of the N-terminal catalytic domain whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. We propose that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment.

scholarly journals Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization

2011 ◽  
Vol 301 (3) ◽  
pp. F554-F564 ◽  
Author(s):  
Sierra Delarosa ◽  
Julie Guillemette ◽  
Joan Papillon ◽  
Ying-Shan Han ◽  
Arnold S. Kristof ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Gel Filtration ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
Fk506 Binding Protein ◽  
Induced Apoptosis ◽  
Injury And Repair

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1–373) and a modified FK506 binding protein, Fv (Fv-SLK 1–373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1–373. In an in vitro kinase assay, the dimeric Fv-SLK 1–373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1–373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1–373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1–373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

scholarly journals PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum

2000 ◽  
Vol 347 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Valerie BRACCHI-RICARD ◽  
Sailen BARIK ◽  
Cherie DELVECCHIO ◽  
Christian DOERIG ◽  
Ratna CHAKRABARTI ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Sequence Similarity ◽  
Small Subunit ◽  
Mitogen Activated Protein Kinase ◽  
Cdna Clones ◽  
Cyclin Dependent Kinase ◽  
Mitogen Activated Protein

We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T173PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2+ ions as a cofactor. The Lys38 → Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.

Role of Amplified Genes in the Production of Autoantibodies

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2158-2166 ◽  
Author(s):  
Nicole Brass ◽  
Alexander Rácz ◽  
Christine Bauer ◽  
Dirk Heckel ◽  
Gerhard Sybrecht ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Squamous Cell ◽  
Lung Carcinoma ◽  
Chromosomal Abnormalities ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Squamous Cell Lung Carcinoma ◽  
Expression Library ◽  
Cell Lung Carcinoma

Abstract A variety of previously published studies have shown the presence of autoantibodies directed against oncogenic proteins in the sera of patients with tumors. Generally the underlying genetic aberration responsible for the induction of an immune response directed against an abnormal protein is unknown. In our studies we analyzed the role of gene amplification in the production of autoantibodies in squamous cell lung carcinoma. We screened a cDNA expression library with autologous patient serum and characterized the isolated cDNA clones encoding tumor expressed antigens termed LCEA (lung carcinoma expressed antigens). As determined by sequence analysis, the 35 identified cDNA clones represent 19 different genes of both known and unknown function. The spectrum of different clones were mapped by polymerase chain reaction (PCR) and fluorescence in-situ hybridization, showing that a majority are located on chromosome 3, which is frequently affected by chromosomal abnormalities in lung cancer. Gene amplification of 14 genes was analyzed by comparative PCR. Nine genes (65% of all analyzed genes) were found to be amplified; furthermore, most of them are also overrepresented in the pool of cDNA clones, suggesting an overexpression in the corresponding tumor. These results strongly suggest that gene amplification is one possible mechanism for the expression of immunoreactive antigens in squamous cell lung carcinoma.

scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals NuMA: an unusually long coiled-coil related protein in the mammalian nucleus.

1992 ◽  
Vol 116 (6) ◽  
pp. 1303-1317 ◽  
Author(s):  
C H Yang ◽  
E J Lambie ◽  
M Snyder
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Early Prophase ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mitotic Apparatus ◽  
Reading Frame ◽  
Nuclear Lamins

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.

scholarly journals Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38)

1990 ◽  
Vol 270 (1) ◽  
pp. 97-102 ◽  
Author(s):  
J P Luzio ◽  
B Brake ◽  
G Banting ◽  
K E Howell ◽  
P Braghetta ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Polyclonal Antiserum ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Trans Golgi Network ◽  
Sequencing And Expression ◽  
Novel Strategy ◽  
Golgi Network

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network.

Sequence, expression and localization of calmodulin-domain protein kinases inEimeria tenellaandEimeria maxima

Parasitology ◽  
1996 ◽  
Vol 113 (5) ◽  
pp. 439-448 ◽  
Author(s):  
P. P. J. Dunn ◽  
J. M. Bumstead ◽  
F. M. Tomley
Keyword(s):  
Host Cell ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Sequence Data ◽  
Host Cells ◽  
Cdna Clones ◽  
Host Cell Membrane ◽  
Amino Terminal ◽  
Domain Protein

SUMMARYWe have isolated and sequenced cDNA clones fromEimeria tenellaandEimeria maximawhich encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of theE. tenellacalmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells inin vitroculture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells byE. tenella.

scholarly journals Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.

1993 ◽  
Vol 123 (2) ◽  
pp. 357-371 ◽  
Author(s):  
D Masson ◽  
T E Kreis
Keyword(s):  
Epithelial Cells ◽  
Molecular Characterization ◽  
Hela Cells ◽  
Binding Proteins ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Microtubule Binding ◽  
Terminal Domain

A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.

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