Tcl-30, a new T cell-specific gene expressed in immature glucocorticoid-sensitive thymocytes.

Glucocorticoid treatment induces programmed cell death (apoptosis) in susceptible T lymphocytes. We have previously isolated and characterized 13 genes induced by agents that cause apoptosis in the murine thymoma cell line, WEHI-7TG. One of these genes, now designated Tcl-30, encodes a 2.4-kb mRNA that is specifically expressed in thymus. Sequence analysis of a full-length cDNA for Tcl-30 reveals a potential open reading frame of 454 amino acids that shares sequence identity to a human placental-specific protein of unknown function, PP11. The putative protein encoded by Tcl-30 also contains a cysteine-rich somatomedin B-like domain found in vitronectin, PP11, and the plasma cell membrane glycoprotein, PC-1. Subpopulations of murine thymocytes sorted on the basis of their expression of the CD4, CD8, and surface heat-stable Ag (HSA) were analyzed by an RNase protection assay to determine the expression of Tcl-30 as a function of T cell development. Tcl-30 was expressed exclusively in the HSA+ T cell populations (CD4-CD8-HSA+; CD4+CD8-HSA+; CD4-CD8+HSA+; and CD4+CD8+HSA+) and not in the HSA- single positive T cell populations (CD4+CD8- or CD4-CD8+) of the thymus or spleen. Therefore, we conclude that Tcl-30 expression is lost during T cell maturation and is absent at the most mature stages of T cell development. The function of Tcl-30 is unknown; however, the CD4+CD8+ double-positive subpopulation expressing Tcl-30 represents thymocytes destined to undergo massive intrathymic cell death. The possibility that Tcl-30 expression may define a population of T lymphocytes that is sensitive to glucocorticoid-mediated apoptosis is discussed.     

Homology of placental protein 11 and pea seed albumin 2 with vitronectin.

Vitronectin (complement S-protein), a plasma and tissue glycoprotein of 75 kDa, shares the amino-terminal somatomedin B domain with the membrane glycoprotein PC1 of plasma cells and several hemopexin-type repeats with hemopexin and the non-catalytic carboxy-terminal domain of collagenases. It serves as a ligand for certain integrin receptors, binds to distinct members of the serpin family and inhibits the pore-forming cytolytic reaction of the terminal complement pathway. Computer-assisted data base searches revealed the presence of a single somatomedin B domain in the recently cloned placental protein 11, and four hemopexin-type repeats in the cytosolic plant protein PA2, the major albumin of pea seeds, whose function is unknown. Our finding shows that hemopexin-type repeats are present in extracellular as well as in cytosolic proteins and most likely originated before the divergence of the animal and plant kindoms.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues.

A thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs. These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE. Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.     

Novel organization and processing of the guinea pig pancreatic polypeptide precursor

Studies on New World hystricomorph rodents have revealed interesting structural divergences in the peptide hormones of the islets of Langerhans, particularly with respect to insulin and glucagon. Herein we report the isolation and sequencing of a cDNA encoding the precursor of pancreatic polypeptide (PP) from a guinea pig pancreas cDNA library. The 126-residue precursor sequence is predicted to include a 26-residue NH2-terminal signal peptide followed by the 36-amino acid PP hormonal sequence and a large COOH-terminal extension. The sequence identity between guinea pig and human PP is 89% (32/36 residues), and the predicted sequence is in agreement with that reported by Eng et al. (Eng, J., Huang, C.-G., Pan, Y.-C. E., Hulmes, J. D., and Yalow, R. S. (1987) Peptides 8, 165-168). In contrast, the icosapeptide domain in the guinea pig precursor exhibits only 40% (8/20) identity with the corresponding human precursor domain, and the COOH-terminal extension differs greatly in both sequence and size. The guinea pig precursor lacks the monobasic processing site (Pro-Arg) found at the COOH terminus of the icosapeptide domain in human, ovine, canine, and feline proPP. An icosapeptide is thus not likely to be liberated as such from this precursor. Of particular interest in guinea pig proPP is the substitution of serine for arginine at the dibasic amino acid processing site on the COOH-terminal side of the PP domain. Results of radioimmunoassays of gel-filtered protein fractions from a guinea pig pancreas extract indicate that efficient proteolytic cleavage takes place at this Lys-Ser site and that mature guinea pig PP is normally carboxyamidated.     

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.     

Cloning of a urochordate cDNA featuring mammalian short consensus repeats (SCR) of complement-control protein superfamily

Mammalian tumor necrosis factor (TNF)-α degenerate polymerase chain reaction (PCR) primers were used to amplify a probe from Botryllus schlosseri (colonial ascidian) allogeneic rejection-cDNA library. A PCR product (269 bp) was cloned and sequenced encoding an open reading frame (ORF) of 89 amino acids (aa). This clone, which revealed no similarity to TNF-α, but a substantial similarity to mammalian proteins featuring short consensus repeats (SCRs) of the complement control superfamily, was used to probe the rejection-cDNA library. Two partial cDNA clones were isolated and sequenced (Bs. 1, 846 bp; Bs.2, 712 bp). The longest ORF in clone Bs. 1 (which lacks the 5' end of the cDNA) predicts a protein of 251 aa, which differs from Bs.2 at six nucleotides and four aa. We compare the as similarity (up to 50.5%) of Bs.l with the SCR-region of mammalian complement factor H, apolipoprotein H, selectins, and complement receptors type 1 and type 2. A somatomedin B-like domain at the C-terminus of Bs. 1 deduced protein was also recorded. We propose that this mosaic and polymorphic botryllid sequence, featuring mammalian-like SCRs, might be an ancestral molecule in the evolution of the chordate's complement-control protein superfamily.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.     

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.     

Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum. Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum. The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library. Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids. Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum. Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm. This result indicates that PP11 can be synthesized in E. coli and is process by removal of the hydrophobic signal sequence. Both the placental and the processed recombinant PP11 protein exhibit a protease activity.     

The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein

The complete sequence of the human Duchenne muscular dystrophy (DMD) cDNA has been determined. The 3685 encoded amino acids of the protein product, dystrophin, can be separated into four domains. The 240 amino acid N-terminal domain has been shown to be conserved with the actin-binding domain of alpha-actinin. A large second domain is predicted to be rod-shaped and formed by the succession of 25 triple-helical segments similar to the repeat domains of spectrin. The repeat segment is followed by a cysteine-rich segment that is similar in part to the entire COOH domain of the Dictyostelium alpha-actinin, while the 420 amino acid C-terminal domain of dystrophin does not show any similarity to previously reported proteins. The functional significance of some of the domains is addressed relative to the phenotypic characteristics of some Becker muscular dystrophy patients. Dystrophin shares many features with the cytoskeletal protein spectrin and alpha-actinin and is a large structural protein that is likely to adopt a rod shape about 150 nm in length.     

The MyD116 African swine fever virus homologue interacts with the catalytic subunit of protein phosphatase 1 and activates its phosphatase activity

The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.     

绿盲蝽表皮蛋白基因AlCP17的克隆、多克隆抗体制备及表达谱分析

【目的】本研究旨在通过克隆绿盲蝽Apolygus lucorum表皮蛋白(cuticular protein, CP)基因AlCP17、制备其多克隆抗体和分析其时空表达特性,为AlCP17蛋白功能的研究奠定基础,也为进一步解析绿盲蝽表皮发育过程中的信号通路图谱提供依据。【方法】基于前期转录组数据克隆绿盲蝽AlCP17的cDNA全长序列并进行生物信息学分析;构建AlCP17原核表达载体pCZN1-AlCP17并转化大肠杆菌Escherichia coli进行体外表达,利用纯化后的重组蛋白制备AlCP17蛋白多克隆抗体;qRT-PCR检测AlCP17在绿盲蝽不同发育阶段(1-5龄若虫和成虫)和3龄初期若虫不同组织(头、胸、足、中肠、脂肪体和表皮)中的表达谱。【结果】克隆获得绿盲蝽AlCP17全长cDNA序列(GenBank登录号：OM302231),其开放阅读框长594 bp,编码197个氨基酸,预测蛋白分子量为21.69 kD,理论等电点为6.11。编码的蛋白质具有典型的表皮蛋白结构特征,含有节肢动物表皮蛋白几丁质保守结合域(non-cysCBD),即Chitin＿bind＿4结构域;氨...     

Cell cycle-dependent expression and centrosome localization of a third human aurora/Ipl1-related protein kinase, AIK3

We earlier isolated cDNAs encoding novel human protein kinases AIK and AIK2 sharing high amino acid sequence identities with Drosophila Aurora and Saccharomyces cerevisiae Ipl1 kinases whose mutations cause abnormal chromosome segregation. In the present study, a third human cDNA (AIK3) highly homologous to aurora/IPL1 was isolated, and the nucleotide sequence was determined. This cDNA encodes 309 amino acids with a predicted molecular mass of 35.9 kDa. C-terminal kinase domain of AIK3 protein shares high amino acid sequence identities with those of Aurora/Ipl1 family protein kinases including human AIK, human AIK2, Xenopus pEg2, Drosophila Aurora, and yeast Ipl1, whereas the N-terminal domain of AIK3 protein shares little homology with any other Aurora/Ipl1 family members. AIK3 gene was assigned to human chromosome 19q13.43, which is a frequently deleted or rearranged region in several tumor tissues, by fluorescence in situ hybridization, somatic cell hybrid panel, and radiation hybrid cell panel. Northern blot analyses revealed that AIK3 expression was limited to testis. The expression levels of AIK3 in several cancer cell lines were elevated severalfold compared with normal fibroblasts. In HeLa cells, the endogenous AIK3 protein level is low in G1/S, accumulates during G2/M, and reduces after mitosis. Immunofluorescence studies using a specific antibody have shown that AIK3 is localized to centrosome during mitosis from anaphase to cytokinesis. These results suggest that AIK3 may play a role(s) in centrosome function at later stages of mitosis.     

Identification and characterization of a novel Golgi protein, golgin-67

In the course of screening a lambdagt11 human leukemic T-cell cDNA expression library with an antibody specific to the mitotic target of Src, Sam68, we identified and cloned a cDNA encoding a novel protein with a predicted molecular mass of 51.4 kDa. Polyclonal antibodies raised to a His(6)-tagged construct of this protein, detected a approximately 67-kDa protein in immunoprecipitation experiments, and cytological studies showed that this protein localized to the Golgi complex, through colocalization experiments with specific Golgi markers. Therefore, we designated this protein golgin-67. Sequence analysis revealed that golgin-67 is a highly coiled-coil protein, with potential Cdc2 and Src kinase phosphorylation motifs. It has sequence homologies to other Golgi proteins, including the coatamer complex I vesicle docking protein, GM130. Structurally, golgin-67 resembles, golgin-84, an integral membrane Golgi protein with an N-terminal coiled-coil domain and a single C-terminal transmembrane domain. The C-terminal region of golgin-67, which contains a predicted transmembrane domain, was demonstrated to be essential for its Golgi localization.     

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.