Biomedical subjects

I Dulubova

Publications and source records attributed to I Dulubova.

10 recordsLinked to original sources

PubMed2001

Functional analysis of conserved structural elements in yeast syntaxin Vam3p.

Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.

Conserved Sequence↗

PubMed2001

Vam3p structure reveals conserved and divergent properties of syntaxins.

Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion.

Amino Acid Sequence↗

PubMed2001

ARPP-16/ARPP-19: a highly conserved family of cAMP-regulated phosphoproteins.

ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.

Amino Acid Sequence↗

PubMed1999

A conformational switch in syntaxin during exocytosis: role of munc18.

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

Animals↗

PubMed1999

Regulation of Na+, K+-ATPase isoforms in rat neostriatum by dopamine and protein kinase C.

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.

Animals↗

PubMed1998

Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A.

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.

Animals↗

PubMed1997

Mutation of the protein kinase C phosphorylation site on rat alpha1 Na+,K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness.

The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that is of vital importance for functioning of all eukaryotic cells. Na+, K+-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and these sites of phosphorylation have been identified. In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na+,K+-ATPase. In COS cells transfected with wild type rat Na+,K+-ATPase alpha1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alpha1 in which the PKC phosphorylation site, Ser-23, had been mutated into alanine. Phorbol dibutyrate inhibited Na+,K+-ATPase-dependent ATP hydrolysis and Rb+ uptake in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Cells expressing the S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na+, K+-ATPase alpha1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha1 and suggest a connection between this enzyme and cell adhesion.

Animals↗

PubMed1996

Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

Amino Acid Sequence↗

PubMed1993

Cloning and structural analysis of alpha-latroinsectotoxin cDNA. Abundance of ankyrin-like repeats.

alpha-Latroinsectotoxin (alpha-LIT), purified from venom glands of the black widow spider Latrodectus mactans tredecimguttatus, is a presynaptic neurotoxin selective only for insects. A cDNA encoding the putative alpha-LIT precursor was isolated from a spider venom gland cDNA library. The cDNA contains a 4236-base-pair open reading frame corresponding to a 157826-Da protein composed of 1411 amino acids. The mature alpha-LIT, with molecular mass approximately 130 kDa, is probably derived from double processing in the N-terminal and C-terminal regions of the primary translation product. The structure region, extending over residues 464-1176, is composed almost entirely of ankyrin-like repeats which represent a motif also found in the alpha-latrotoxin (alpha-LTX), which has selective action on vertebrates. Total alignment of the alpha-LIT and alpha-LTX amino acid sequences reveals an overall similarity of 34.1%. Strong sequence divergence is observed in analogous cysteine-rich regions situated within the ankyrin-repeat domains of both alpha-LIT and alpha-LTX.

Amino Acid Sequence↗

PubMed1992

Structure of the low molecular weight protein copurified with alpha-latrotoxin.

Some samples of latrotoxin purified from the black widow spider venom contain two components: alpha-latrotoxin (M(r) approximately 130,000) and a low mol. wt protein with M(r) about 8000. Clones carrying the cDNA sequence for the low mol. wt protein copurified with alpha-latrotoxin were isolated from spider venom glands. Nucleotide sequence analysis of the cloned cDNA revealed the primary structure of the polypeptide to be 18 amino acids signal peptide and 70 amino acids protein chain with mol. wt of 7947 and pI of approximately 4.0. The protein exhibits certain structural homology with erabutoxin-a from the sea snake.

Amino Acid Sequence↗