cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization.

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.     

Vesicular reuptake inhibition by a synaptotagmin I C2B domain antibody at the squid giant synapse

Synaptotagmin (Syt) I, a ubiquitous synaptic vesicle protein, comprises a transmembrane region and two C2 domains. The C2 domains, which have been shown to be essential for both synaptic vesicle exocytosis and endocytosis, are also seen as the Ca(2+) sensors in synaptic vesicular release. In a previous study, we reported that a polyclonal antibody raised against the squid (Loligo pealei) Syt I C2B domain, while inhibiting vesicular endocytosis, was synaptic release neutral at the squid giant synapse. Recent reports concerning the C2B requirements for synaptic release prompted us to readdress the role of C2B in squid giant synapse function. Presynaptic injection of another anti-Syt I-C2B antibody (using recombinant whole C2B domain expressed in mammalian cell culture as an antigen) into the presynaptic terminal reproduced our previous results, i.e., reduction of vesicular endocytosis without affecting synaptic release. This set of results addresses the issue of the geometrical arrangement of the Ca(2+) sensor, allowing the C2B domain antibody to restrict Ca(2+)-dependent C2B self-oligomerization without modifying the Ca(2+)-dependent release process.     

The involvement of neurofilament heavy chain phosphorylation in the maturation and degeneration of rat oocytes

Neurofilaments (NF) are intermediate filament proteins that were originally found to be expressed in neurons and are involved in the maintenance of axonal structure. However, there has not been much research on the expression and physiological significance of NF in other organs. In the present study, we examined the expression of NF in rat ovaries and found that NF heavy chain (NF-H) was expressed in oocytes of follicles from the primary to mature stages, ovulated oocytes, fertilized zygotes, and degenerative oocytes of atretic follicles. Cytoplasmic NF-H disappeared at the two-cell stage of embryonic development, whereas degenerative oocytes of atretic follicles continued to express NF-H even after fragmentation. An antibody that specifically recognizes phosphorylated NF-H (pNF-H) was used to examine the pattern of NF-H phosphorylation in oocytes. pNF-H was detected in the cytoplasm and nuclei of oocytes of mature and atretic follicles, ovulated oocytes, and one-cell zygotes. Treatment with 3,3'-iminodipropionitrile, which induces aberrant phosphorylation of NF in the perikarya of neurons and causes neuropathy, induced oocyte degeneration with follicular atresia, phosphorylation of NF-H in oocytes, and ovarian gene expression of cyclin-dependent kinase 5, a candidate kinase of NF-H. However, an indicator of neuron degeneration, Fluoro-Jade C, failed to stain the pNF-H-immunopositive oocytes. Our results indicate that NF-H expressed in oocytes may be involved in the maintenance of oocyte structure during follicular growth and that the phosphorylation of NF-H in ephemeral oocytes may contribute to the degeneration process of oocytes.     

Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.     

A conserved domain is present in different families of vesicular fusion proteins: A new superfamily

We have analyzed conserved domains in t-SNAREs [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors in the target membrane], proteins that are believed to be involved in the fusion of transport vesicles with their target membrane. By using a sensitive computer method, the generalized profile method, we were able to identify a new homology domain that is common in the two protein families previously identified to act as t-SNAREs, the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) families, which therefore constitute a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain was also identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode, and mammalian proteins were identified that belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins.     

Resolution and purification of a neurofilament-specific kinase.

Both in vivo and in vitro, neurofilaments (NFs) are among the most highly phosphorylated proteins known. The majority of the NF phosphorylation sites reside on the carboxyl-terminal tails of the proteins. We have isolated and characterized an effector-independent neurofilament-specific protein kinase from bovine spinal cord that is associated with the NF complex and exhibits a marked substrate specificity for NF-H, the largest subunit of the NF triplet. This kinase activity emerges from a NF-conjugated affinity column coincident with a 67-kDa doublet on NaDodSO4/polyacrylamide gels and has a purity of greater than 90%. The purified enzyme exclusively phosphorylates NF-H tails and is dependent on prior phosphorylation of this molecule. The enzyme is also not autophosphorylated. While the molecular properties and substrate specificities of the NF kinase distinguish it from cAMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin kinase, and casein kinases I and II, it exhibits certain properties similar to, but different from, the growth-associated histone H1 kinase. The molecular properties and specific sequence requirements of the NF kinase suggest that this enzyme could play a pivotal role in the phosphorylation of NFs in normal and pathological states such as Alzheimer disease, where NFs are hyperphosphorylated.     

Domain structure of a mammalian myosin I beta.

We have determined the primary structure of a myosin I (called mammalian myosin I beta, MMI beta) from bovine brain and identified its functional domains. The protein was previously purified from brain and adrenal gland. Several constructs were generated and expressed in Escherichia coli as glutathione S-transferase fusion proteins and the recombinant proteins were recognized by monoclonal antibodies that recognize either "head" or "tail" domains of native myosin I. A gel overlay method was used to confirm that calmodulin binds to the consensus calmodulin-binding sequence in MMI beta. Binding assays were used to detect interaction with anionic phospholipid vesicles. We conclude that MMI beta consists of an amino-terminal 80.5-kDa domain that contains the ATP- and actin-binding sites, followed by an 8.5-kDa domain with three calmodulin-binding sequences and a basic 30-kDa carboxyl-terminal tail segment that binds to anionic phospholipids and membranes.     

A conserved double-stranded RNA-binding domain.

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.     

Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions.

NF1 was first identified as the gene responsible for the pathogenesis of the human genetic disorder neurofibromatosis type 1. cDNA cloning revealed that its putative protein product has a domain showing significant sequence homology with the mammalian Ras GTPase activating protein and two yeast Saccharomyces cerevisiae proteins, Ira1 and Ira2. The Ras GTPase activating protein-related domain of the NF1 gene product (NF1-GRD) stimulates GTPase activity of normal Ras proteins but not of oncogenic mutant Ras from both mammalian and yeast cells. Thus, in yeast, NF1-GRD can suppress the heat-shock-sensitive phenotype of ira- cells but not the same phenotype of activated RAS such as RAS2Val19 and RAS2Leu68. We have screened a pool of mutagenized NF1 expression plasmids and obtained two mutant NF1 cDNA clones that can suppress the heat-shock-sensitive phenotype of RAS2Val19 cells. One clone (NF201) suppressed RAS2Leu68, RAS2Ser41, and RAS2Val19, whereas another clone (NF204) preferentially suppressed RAS2Val19. When expressed in mammalian cells, these mutant NF1-GRDs were able to induce the morphological reversion of v-ras-transformed NIH 3T3 cells. Both wild-type and mutant NF1-GRDs can stimulate the GTPase activity of normal but not transforming Ras. We suggest that mutant NF1-GRDs may bind tightly to transforming Ras, which stays in GTP-bound conformation, thus preventing the interaction with the putative effector molecule. On the other hand, normal Ras cannot be sequestered since the bound GTP is rapidly hydrolyzed upon interaction with mutant NF1-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled.     

Identification of the major multiphosphorylation site in mammalian neurofilaments.

The sequence Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neurofilament (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs)--i.e., MAP2 and tau.     

Neurofilament and Intermediate Filament Immunoreactivity in Human Intestinal Myenteric Neurons

It has been reported previously that ratmyenteric neurons have neurofilament (NF)immunoreactivity that differs from the brain. Now theresult of a study of neurofilaments and intermediatefilament immunoreactivity in human colon and ileum using a panel ofantibodies and indirect immunofluorescence techniques isreported here. Results with polyclonal neurofilamentantisera showed positive immunoreactivity in subsets of myenteric neurons. Results with peripherinand alpha-internexin showed immunoreactivity in someneurons that contained neurofilaments and in many thatwere neurofilament negative, similar to our observations in rat. Some monoclonal antibodies to epitopeson NF-M and NF-H demonstrated weak or negativeimmunoreactivity in human myenteric neurons yet showedpositive immunoreactivity in brain. Some of theseantibodies are phosphorylation dependent, suggesting NF-Mand NF-H epitopes in myenteric neurons are not asphosphorylated as in brain; other antibodies arephosphorylation independent, suggesting otherdifferences or masking of epitopes. In summary,neurofilaments are present in a subset of myentericneurons. In those human myenteric neurons that containthem, the neurofilaments appear immunologically distinctfrom those in the brain.     

Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors.

The mammalian sex-determining gene SRY (sex-determining region on Y chromosome) encodes a member of the high mobility group (HMG) family of regulatory proteins. The HMG domain of the SRY protein represents a DNA binding motif that displays rather unusually weak evolutionary conservation of amino acids between human and mouse sequences. Together with the previous finding that the human (h) SRY gene is unable to induce a male phenotype in genetically female transgenic mice, these observations raise questions concerning the DNA binding properties of SRY proteins. Here, we present data that indicate that the DNA binding and bending properties of the HMG domains of murine (m) SRY and hSRY differ from each other. In comparison, mSRY shows more-extensive major-groove contacts with DNA and a higher specificity of sequence recognition than hSRY. Moreover, the extent of protein-induced DNA bending differs from the HMG domains of hSRY and mSRY. These differences in DNA binding by hSRY and mSRY may, in part, account for the functional differences observed with these gene products.     

Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin beta tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirmed that these interactions are canonical PTB domain-ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin beta tail-PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism.     

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution.

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.     

Melatonin protects SH-SY5Y neuroblastoma cells from calyculin A-induced neurofilament impairment and neurotoxicity

Hyperphosphorylation of cytoskeletal proteins seen in Alzheimer's disease is most probably the result of an imbalanced regulation in protein kinases and protein phosphatases (PP) in the affected neurons. Previous studies have revealed that PP-2A and PP-1 play important roles in the pathogenesis. Employing human neuroblastoma cells, we found that 10 nM calyculin A (CA), a selective inhibitor of PP-2A and PP-1, significantly increased phosphorylation and accumulation of neurofilament (NF) in the cells. Levels of NF-M (middle chain) and NF-L (light chain) mRNA decreased after CA treatment. Additionally, CA led to a decreased cell viability determined by MTT and crystal violet assay. Melatonin efficiently protects the cell from CA-induced alterations in NF hyperphosphorylation and accumulation, suppressed NF gene expression as well as decreased cell viability. It is concluded that inhibition of PP-2A/PP-1 by CA induces abnormalities in NF metabolism and cell survival, and melatonin efficiently arrests the lesions.     

PX domain takes shape

In recent years, a number of protein domains have been identified that bind phosphoinositides and direct proteins to membrane targets. A recent addition to this group is the Phox homology or PX domain, a 120-amino acid domain conserved from yeast to humans, which is present in proteins involved in cell signaling, protein sorting, vesicle fusion, and the assembly of components of the superoxide generating system of neutrophils. These domains have varying affinities for phosphatidylinositol-3-phosphate (PI(3)P), and PI(3,4) and (4,5) bisphosphates, which couple the PI kinase and phosphatase signaling networks to the assembly of proteins at membrane surfaces. These PX domains also contain a PXXP motif, allowing them to bind to proteins containing Src homology 3 (SH3) domains.     

弓形虫感染对大鼠脑组织神经丝mRNA表达的影响

目的探讨弓形虫感染对大鼠脑组织神经丝（NF）mRNA表达和细胞免疫水平的影响。方法将4周龄雄性SD大鼠随机分成2组：弓形虫感染组（A组）腹腔感染弓形虫速殖子2×10^2／ml悬液2ml;正常对照组（B组）腹腔注射灭菌生理盐水2ml。感染弓形虫速殖子9周后,应用逆转录聚合酶链反应检测大鼠大脑组织中高分子量NF（NF—H）、中分子量NF（NF—M）和低分子量NF（NF—L）mRNA表达水平;流式细胞术检测大鼠外周血CD3^＋、CD4^＋、CD8^＋T淋巴细胞;ELISA测定其血清IFN-γ、TNF-α、IL-4等细胞因子。结果大鼠弓形虫感染9周后,大脑组织NF．LmRNA下降为正常对照组的64％（P〈0．01）;NF—M为96％（P〉0．05）;NF—H为89％（P〈0．05）。感染组大鼠的CD4^＋和CD8^＋T淋巴细胞与正常对照组相比,差异均无统计学意义（P均〉0．05）;感染组大鼠血清中IFN-γ、TNF-α、IL-4等细胞因子的水平均高于正常对照组大鼠水平（P〈0．05）。结论弓形虫感染可导致大鼠脑组织中NF亚单位mRNA表达水平的下降,血清IFN-γ、TNF-α、IL-4水平的升高。     

Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca(2+)-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca(2+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca(2+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.