Localization of the linker domain of Ca2+/calmodulin-dependent protein kinase II.

Electron micrographs of rotary shadowed replicas of alpha-Ca2+/calmodulin-dependent protein kinase II reveal a flower-shaped multimeric molecule with a central particle surrounded by 8-10 smaller peripheral particles. Peripheral particles are attached to the central particle by thin arms or "linkers." Movement of peripheral particles to contact each other for autophosphorylation is likely to involve these linkers. It has generally been accepted that the segment 317-328 of the alpha-subunit constitutes the linker domain. In the present study we test this assumption by generating a mutant lacking the proposed sequence. The mutant has biochemical and morphological properties similar to those of the wild type, and a thin linker is occasionally observed in replicas from either type. The results indicate that the deleted sequence does not correspond to the linker domain. This conclusion, combined with observations from two recent studies which identify the C-terminal domain involved in oligomerization, narrows down the location of the linker domain within the sequence 330-354.

Kinesin light chains are essential for axonal transport in Drosophila.

Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.

Kinesin light chain in a eubacterium.

A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum. Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats. Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis. Attempts to identify a canonical kinesin heavy-chain gene or protein were unsuccessful, suggesting that a kinesin heavy chain may be absent or unnecessary for kinesin light-chain function in this eubacterium. Our findings establish that certain basal elements of eukaryotic cellular transport appear to be resident in eubacteria. We discuss the possibility that the eukaryotic kinesin light chain was acquired by lateral gene transfer.

Calexcitin: a signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability.

A previously uncharacterized 22-kDa Ca(2+)-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca2+, calexcitin bound GTP and possessed GTPase activity. Calexictin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca2+ and the absence of endogenous Ca2+/calmodulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 +/- 6 pS K+ channels. Calexcitin thus appears to directly regulate K+ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca(2+)-activated signaling molecule that increases cellular excitability, which would in turn increase Ca2+ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca2+.

Application of a membrane fusion assay for rapid drug screening.

PURPOSE: The purpose of this study is to develop an in vitro assay for screening drug and their effects on membrane fusion and lysis of intracellular organelles. METHODS: A 96-well microtiter-dish turbidimetric assay using membrane components of the eggs of sea urchins, a marine invertebrate, was applied to monitor granule fusion and/or lysis. RESULTS: Of 18 drugs screened, 16 had no effect. One antineoplastic drug, tamoxifen, disrupted intracellular membranes in a calcium independent manner. Taxol, another antineoplastic drug, specifically inhibited calcium triggered exocytosis. CONCLUSIONS: This assay is inexpensive, simple, rapid, and does not require the sacrifice of animal life. It has the potential to identify drugs that are membrane active, as well as those which specifically perturb events involved in the secretion process.

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5.

Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

cAMP-dependent phosphorylation of Aplysia twitchin may mediate modulation of muscle contractions by neuropeptide cotransmitters.

Acting through a cAMP-cAMP-dependent protein kinase (cAPK) cascade, members of two neuropeptide families, the small cardioactive peptides and myomodulins, modulate contraction amplitude and relaxation rate in the accessory radula closer (ARC) muscle of the marine mollusc Aplysia californica. An approximately 750-kDa phosphoprotein was identified in the ARC muscle as the major substrate for cAPK activated either by application of neuropeptides or by peptides released by motorneuron stimulation at physiological frequencies. Immunoblot and immunoelectron microscopy experiments revealed the widespread presence of this protein in Aplysia muscles and its colocalization with contractile filaments in the ARC muscle. Sequence analysis of proteolytic peptide fragments derived from the protein indicated that it is structurally related to the muscle protein twitchin. Finally, the level of neuropeptide-induced phosphorylation of the protein correlated well with peptidergic modulation of the relaxation rate of the muscle. We propose that twitchin in Aplysia, and perhaps in other species, may mediate the modulation of the relaxation rate of muscle contractions.

Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues.

The VAMPs/synaptobrevins (Vp/Sybs) are small integral membrane proteins. Two isoforms, Vp/Syb 1 and Vp/Syb 2, are considered to be specific to neural tissue. They are associated with synaptic vesicles and are believed to play an important role in neurotransmitter release. A third isoform, cellubrevin, has recently been found in non-neural tissues. We now report that the distribution of Vp/Syb 1 and Vp/Syb 2 is wider than previously thought. RNA transcripts for both Vp/Syb 1 and Vp/Syb 2 were found in rat skeletal muscle and in several other rat non-neural tissues, and antibodies specific for Vp/Syb 2 detected a protein in the endoplasmic reticulum-Golgi area of skeletal muscle. Thus Vp/Sybs 1 and 2 are not restricted to the nervous system but appear to be co-expressed with cellubrevin in many different tissues. This redundancy of Vp/Sybs in a single cell may be required to control the specificity of vesicle-target interaction in the several pathways of intracellular vesicle traffic that are operative within each cell.

Kinesin light chains: identification and characterization of a family of proteins from the optic lobe of the squid Loligo pealii.

Multiple transcripts coding for kinesin light chain isoforms are present in the tissues of the squid Loligo pealii. Isoform diversity arises through alternative RNA splicing in the amino and carboxyl termini of the putative proteins. Comparison to rat and Drosophila proteins demonstrates a remarkable conservation of structural domains and regulatory motifs. We have identified a PEST domain that may be the site of degradative uncoupling of kinesin functions. Selective transcript distribution occurs in disparate tissues, suggesting an adaptation toward specialized functions. Expression is highest in the nervous system and some evidence for neural-specific transcripts is provided. In neurons, this may relate to the differential targeting of specific membrane-bound organelles such as synaptic vesicles.

The myomodulin-related neuropeptides: characterization of a gene encoding a family of peptide cotransmitters in Aplysia.

The myomodulin-related peptides comprise a family of cotransmitters that modulate neuromuscular signaling in the feeding system of Aplysia. In this study, cDNA clones encoding a myomodulin precursor polypeptide were isolated and characterized. This precursor contains seven different myomodulin-related peptides, one of which, myomodulin A, is present in 10 contiguous copies. The sequence of a myomodulin genomic clone indicates that all of these myomodulin-related peptides are encoded on a single exon. The myomodulin gene is expressed in a tissue-specific manner and myomodulin mRNA is localized to specific neurons in the Aplysia CNS. The presence of multiple related neuropeptides can greatly increase the range and precision of signaling at synapses where they act as modulator cotransmitters.

The buccalin-related neuropeptides: isolation and characterization of an Aplysia cDNA clone encoding a family of peptide cotransmitters.

The buccalin-related peptides, buccalin A and buccalin B, are members of a family of cotransmitters that modulate neuromuscular transmission in Aplysia. In this study, a third buccalin-related peptide, buccalin C, was purified from neuronal elements in the accessory radula closer, a muscle involved in the animal's feeding behavior. Oligonucleotide probes based upon the amino acid sequence of buccalin C were used to isolate cDNA clones that encode a buccalin precursor polypeptide. The buccalin precursor contains 19 distinct buccalin-related peptides, several of which are present in multiple copies. The buccalin gene appears to be present in a single copy, with one allele containing a small insert. Expression of this gene occurs in a tissue-specific manner and mRNA transcripts are abundant within neurons in the Aplysia CNS. This large family of neuropeptides may exert extraordinarily complex modulatory actions at synapses where they serve as cotransmitters.

Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini.

Previously, two forms of cAMP-dependent protein kinase catalytic subunit generated by mutually exclusive use of two internal exon cassettes (A1 and A2) were demonstrated in Aplysia neurons. Here, it is shown that there also exist catalytic subunits with alternative N termini derived from two exons, N1 and N2, expressed in combination with either of the internal cassettes. Processed transcripts including N1 or N2 sequences are of about equal abundance in the nervous system, arise through alternative promoter use, and encode catalytically active polypeptides. The N2 amino acid sequence is 21 residues longer than the N1 sequence and is homologous to the nonmyristoylated N terminus of the TPK1 gene product, a yeast catalytic subunit homolog. These data support the view that cAMP-dependent protein kinase activity in Aplysia neurons is produced by a complex array of regulatory and catalytic subunits that generate multiple holoenzymes with a spectrum of properties.

A regulatory subunit of the cAMP-dependent protein kinase down-regulated in aplysia sensory neurons during long-term sensitization.

Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation. To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI. Immunoblots with antisera raised against the R protein overexpressed in E. coli show that the diminished binding activity, which occurs in long-term facilitation, results from coordinate loss of R protein isoforms. No change was detected in the amount of transcripts for R subunits, suggesting that the down-regulation results from enhanced proteolytic turnover.

Cloning and characterization of Ca(2+)-dependent and Ca(2+)-independent PKCs expressed in Aplysia sensory cells.

We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)-independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones. Sacktor and Schwartz (1990) measured the proportion of kinase activity that can be translocated to membrane in Aplysia sensory neurons and ganglia by stimuli that produce the presynaptic facilitation underlying behavioral sensitization. Much less Apl I and Apl II are translocated, suggesting that still other isoforms of PKC exist in these cells.

Primary structure of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified from the ovotestis of the marine mollusk Aplysia californica. The enzyme converts NAD to cyclic ADP-ribose (cADPR), which is a potent mobilizer of Ca2+. It is likely that the NADase serves to raise Ca2+ levels in the ova at appropriate times. A 1.2-kb cDNA clone containing the complete coding sequence of the native NADase protein was isolated from an unamplified ovotestis cDNA library and represents the first cloning of an NADase that generates cADPR. In vitro translation studies indicate that the protein initially has a signal sequence that may help to target it to discrete vesicles of the ova in which it is found. There are 12 cysteines in the open reading frame, two of these being in the signal sequence. No part of the sequence has significant similarity to other proteins or known nucleotide binding site consensus sequences. Northern blot analysis of poly(A)+ selected ovotestis RNA has identified an NADase mRNA of 1.85 kb. In situ hybridization analysis of cryostat sections from ovotestis has shown that the NADase mRNA is restricted to the immature ova, although the NADase protein is present in both immature and mature eggs.

A relative of the catalytic subunit of cyclic AMP-dependent protein kinase in Aplysia spermatozoa.

Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.

Two catalytic subunits of cAMP-dependent protein kinase generated by alternative RNA splicing are expressed in Aplysia neurons.

The amino acid sequences of two catalytic (C) subunits of Aplysia cAMP-dependent protein kinase (cAPK) have been deduced from the nucleotide sequences of cDNAs generated from neuronal poly(A)+ RNA. Both subunits contain 352 residues and are identical except for amino acids 142-183, which differ at 10 out of 42 positions. They derive from alternatively spliced transcripts of a single gene (CAPL) containing two mutually exclusive exon cassettes. CAPL transcripts are present in several classes of identified neurons containing transmitter-sensitive adenylate cyclase, including sensory cells, bag cells, and the left pleural giant cell. Combinatorial expression of the various regulatory (R) and C subunits might produce kinase isoforms with distinct roles in neuronal modulation. Alternatively, holoenzymes with overlapping properties together might contribute to the definition of individual cell types and physiological states.

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the expression of viral RNA and proteins, implying that replication was inhibited at some step between penetration and initiation of genomic functions, perhaps at the stage of uncoating. We therefore examined the possibility that protein kinases and phosphatases, which influence cellular regulation during cAMP-induced differentiation, may be responsible for the phenomenon of coronavirus suppression in oligodendrocytes. Evidence was obtained indicating that normal processing of the phosphorylated nucleocapsid protein is inhibited in differentiated oligodendrocytes, consistent with the notion that JHMV replication might be arrested during uncoating.