Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Cloning and identification of a novel binding protein of CD2 cytoplasmic domain

In order to understand why CD2 has a dual action of transduction of activation or apoptosis signals in T cells under different experimental conditions, we employed a yeast two-hybrid system to look for a binding protein of the cytoplasmic domain of CD2 which may be involved in this issue. A human T cell cDNA library was screened by a cDNA encoding the cytoplasmic domain of CD2 (Thr211-Gln336). The specificity of protein-protein interaction was verified by co-immunoprecipitation. The binding protein obtained, designated CD2cBP, was found to be homologous tov-fos transformation effector protein (Fte-1). As Fte-1 plays a role in cell transformation, growth, protein synthesis and protein-import into mitochondria, this result suggests that CD2cBP may be putatively involved in CD2-mediated signaling.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 10⁶ independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.     

Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide

Many of the physical properties of the erythrocyte membrane appear to depend on the membrane skeleton, which is attached to the membrane through associations with transmembrane proteins. A membrane skeletal protein, protein 4.1, is pivotal in the assembly of the membrane skeleton because of its ability to promote associations between spectrin and actin. Protein 4.1 also binds to the membrane through at least two sites: a high-affinity site on the glycophorins and a site of lower affinity associated with band 3 (ref. 11). The glycophorin-protein 4.1 association has been proposed to be involved in maintenance of cell shape. Here we show that the association between glycophorin and protein 4.1 is regulated by a polyphosphoinositide cofactor. This observation suggests a mechanism which may explain the recently reported dependence of red cell shape on the level of polyphosphoinositides in the membrane.     

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.     

v-Fos transformation effector binds with CD2 cytoplasmic tail

The CD2 molecule, an adhesive and co-stimulating molecule, expresses virtually on all T lymphocytes, thymocytes, as well as natural killer cells, playing an important role in thymus development, T cell activation and apoptosis[1―3]. CD2 molecule consists of an ex- tracellular region with two Ig-like domains, a single membrane-spanning domain, and a positively charged proline-rich cytoplasmic tail, which is highly conserved between different species[1,4―7]. The functions of CD2 in signal t…     

Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.

The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.     

Structural basis for semaphorin signalling through the plexin receptor

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.     

Regulation of glutamate receptor binding by the cytoskeletal protein fodrin

The erythrocyte cytoskeleton, which consists primarily of a meshwork of spectrin and actin, controls cell shape and the disposition of proteins within the membrane. Proteins similar to spectrin have recently been found in diverse cells and tissues, and it is possible that they mediate the capping of cell-surface receptors, although this has not been demonstrated directly. In neurones, the spectrin-like protein fodrin lines the cortical cytoplasm and may link actin filaments to the membrane. Fodrin has been hypothesized to regulate the number of receptor binding sites on neuronal membranes for the putative neurotransmitter L-glutamate. Micromolar calcium concentrations activate the thiol protease calpain I, induce fodrin degradation and more than double the density of glutamate binding sites; these effects are all blocked by thiol protease inhibitors. We have now used specific antibodies to examine further the role of fodrin proteolysis in regulating glutamate receptors. We report that fodrin antibodies block the fodrin degradation and increase in glutamate binding normally induced by calcium, and so provide direct evidence for control of membrane receptors by a non-erythroid spectrin.     

At3g13600截短型蛋白的原核表达载体的构建

拟南芥基因At3g13600编码一种钙调素结合蛋白,序列分析表明,该基因cDNA序列全长1 818 bp,编码一个具有606个氨基酸残基的多肽,推测分子量为68.9 kD;在At3g13600蛋白的N端存在IQ钙调素结合构象.为了从实验上进一步研究该蛋白的钙调素结合特性,通过逆转录聚合酶链式反应( RT-PCR)扩增含...     

Biochemical characterization of the protein encoded by rice osRACD gene

A recombinant plasmid pET-racd was first constructed by cloning osRACD,the development-regulating gene that controls photoperiod fertility transformation in the photoperiod sensitive genic male sterile rice Nongken 58S,into a prokaryote expression vector pET28a(+).It was then transformed into E.Coli BL-21.Cutting with thrombin of the fusion protein extracted from transformants and PAGE separation yielded pure osRACD protein,which was further concentrated using ultrafiltration and renatured using glutathione oxidation/reduction refolding system for later functional study.As demonstrated by in vitro functional assay,the osRACD protein expressed in E.Coli B-21 shows remarkable activity in binding GTP specifically and hydrolyzing it.     

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

Isolation and cloning of a novel cDNA LDBl encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldb1. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldb1, Xenopus Xldb1 and Drosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been named LDB1 (LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts of LDB1 in heart, brain and lung were considerably higher than those in other tissues.