Biomedical subjects

Carl E Creutz

Publications and source records attributed to Carl E Creutz.

7 recordsLinked to original sources

PubMed2005

Interactions of annexins with the mu subunits of the clathrin assembly proteins.

A number of biochemical and genetic studies have suggested that certain annexins play important roles in the endocytic pathway, possibly involving the generation, localization, or fusion of endocytic compartments. In a yeast two-hybrid screen for proteins that interact with the N-terminal domain of annexin A2 we identified the mu2 subunit of the clathrin assembly protein complex AP-2. The interaction depended upon two copies of a Yxx phi amino acid sequence motif (Y = tyrosine, x = variable residue, phi = bulky, hydrophobic residue) in the annexin that is also characteristic of the binding site for mu2 on the cytoplasmic domains of transmembrane receptors. The interaction between mu2 and full-length annexin A2 was demonstrated in vitro to be direct, to require calcium, and to be functional in the sense that annexin A2 was able to recruit the mu2 to immobilized lipids. Examination of other annexins and mu subunits demonstrated that annexin A2 also binds the mu1 subunit of the AP-1 complex, that annexin A6 binds mu1 and mu2, and that annexin A1 binds only mu1. We propose that annexins can "masquerade" as transmembrane receptors when they are attached to membranes in the presence of calcium and that they might therefore function to initiate calcium-regulated coated pit formation at the cell surface or on intracellular organelles.

Amino Acid Sequence↗

PubMed2005

Annexins: linking Ca2+ signalling to membrane dynamics.

Eukaryotic cells contain various Ca(2+)-effector proteins that mediate cellular responses to changes in intracellular Ca(2+) levels. A unique class of these proteins - annexins - can bind to certain membrane phospholipids in a Ca(2+)-dependent manner, providing a link between Ca(2+) signalling and membrane functions. By forming networks on the membrane surface, annexins can function as organizers of membrane domains and membrane-recruitment platforms for proteins with which they interact. These and related properties enable annexins to participate in several otherwise unrelated events that range from membrane dynamics to cell differentiation and migration.

Actins↗

PubMed2004

The tricalbin C2 domains: lipid-binding properties of a novel, synaptotagmin-like yeast protein family.

The tricalbins are a recently discovered family of Saccharomyces cerevisae proteins containing a predicted N-terminal transmembrane domain and at least three C2 domains. They are thought to be yeast homologues of synaptotagmin, a hypothesis supported by structural similarities and past studies that implicated tricalbins in processes of membrane trafficking and sorting. We expressed and purified constructs consisting of single tricalbin C2 domains, and assayed their ability to bind lipids in response to calcium. Protein-lipid overlay assays indicated that the C-terminal C2 domains (C2C) of tricalbins 1 and 3 bind numerous species of acidic phospholipid, including phosphatidylserine and several phosphoinositides, and the amount of protein bound was greatly enhanced in the presence of 1 mM calcium. Sedimentation assays using mixed phosphatidylserine/phosphatidylcholine (PS/PC) vesicles confirmed that the C2C domains of tricalbin 1 and 3 bind membranes in a calcium-responsive manner and showed that they are more sensitive to calcium than the C2A domain of synaptotagmin I. Both assays revealed that all of the C2 domains of tricalbin 2 are insensitive to calcium. Fluorimetric assays exploiting the position of naturally occurring tryptophans in tricalbin 1 C2C and tricalbin 3 C2C confirmed that these domains are capable of binding calcium and that this is coupled to the binding of acidic phospholipid. Combining this with past protein-protein interaction data, we theorize that the calcium-insensitive tricalbin 2 mediates the creation of hetero-oligomeric tricalbin complexes in which tricalbin 1 or 3 or both supply a calcium-dependent membrane binding activity.

Amino Acid Sequence↗

PubMed2004

Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.

The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-kappaB (nuclear factor-kappaB) by TNF-alpha. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa.

Calcium↗

PubMed2003

Structural and dynamic changes in human annexin VI induced by a phosphorylation-mimicking mutation, T356D.

Phosphorylation of some members of the annexin family of proteins may play a significant role in controlling their calcium-dependent interactions with membranes. Recent electron microscopic studies of annexin VI revealed that the protein's two core domains exhibit a great degree of flexibility and are able to undergo a relative conformational change that could potentially initiate contacts between membranes [Avila-Sakar, A. J., et al. (2000) J. Struct. Biol. 130, 54-62]. To assess the possibility of a regulatory role of phosphorylation in this behavior, the crystal structure of a phosphorylation-mimicking mutant (T356D in the flexible connector region of human annexin VI) was determined to 2.65 A resolution. When the mutant is compared to the wild-type annexin VI, subtle differences are seen at the site of the mutation, while larger changes are evident in one of the calcium-binding loops and in the presence of five calcium ions. Furthermore, biochemical studies provide evidence for additional conformational differences between the T356D and wild-type solution structures. Fluorescence emission and acrylamide quenching suggest a higher level of solvent exposure of Trp-343 in the connector region of T356D in the presence of calcium. Comparisons of retardation coefficients in native gel electrophoresis reveal that T356D has a more extended shape, while proteolytic studies show a greater accessibility of a trypsin cleavage site inside the linker region, indicating a conformation more open than the wild-type form. These data provide insights into a possible regulatory mechanism leading to a higher degree of flexibility and possibly a higher calcium binding affinity of annexin VI upon phosphorylation.

Amino Acid Substitution↗

PubMed2003

Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif.

We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins.

Amino Acid Motifs↗

PubMed2002

Purine composition of the crystalline cytoplasmic inclusions of Paramecium tetraurelia.

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.

Animals↗