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Query: UNIPROT:P06889 (Mol)
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We purified a large amount of dynamin with high enzymatical activity from rat brain tissue by a new procedure. Dynamin 0.48 mg was obtained from 20 g of rat brain. The purity of dynamin was almost 98%. Dynamin plays a role of GTPase rather than ATPase. In the absence of microtubules, Michaelis constant (Km) and maximum velocity (Vmax) for dynamin GTPase were 370 microM and 0.25 min-1, respectively, and in their presence, both were significantly accelerated up to 25 microM and 5.5 min-1. On the other hand, the ATPase activity was very low in the absence of microtubules, and even in their presence, Km and Vmax for dynamin ATPase were 0.2 mM and 0.91 min-1. Despite slow GTPase turnover rate in the absence of microtubules, binding of GTP and its nonhydrolizing analogues was very fast, indicating that GTP binding step is not rate limiting. Dynamin did not cause a one-directional consistent microtubule sliding movement just like kinesin or dynein in the presence of 2 mM ATP or 2 mM GTP. We observed the molecular structure of dynamin with low-angle rotary shadowing technique and revealed that the dynamin molecule is globular in shape. Gel filtration assay revealed that these globules were the oligomers of 100-kDa dynamin polypeptide. Dynamin bound to microtubules with a 1:1 approximately 1.2 molar ratio in the absence of GTP. Quick-freeze deep-etch electron microscopy of the dynamin-microtubule complex showed that dynamin decorates the surface of microtubules helically, like a screw bolt, very orderly and tightly with 11.4 +/- 0.9 (SD)nm period. Contrary to the previous report, microtubules make bundles by the attachment of the dynamin helixes around each adjacent microtubule, and no cross-bridge formation was observed.
Mol Biol Cell 1992 Oct
PMID:Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin. 142 74

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.
Plant Mol Biol 1995 Sep
PMID:Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana. 754 29

The VPS1 gene of Saccharomyces cerevisiae encodes an 80-kDa GTPase that associates with Golgi membranes and is required for the sorting of proteins to the yeast vacuole. Vps1p is a member of a growing family of high-molecular-weight GTPases that are found in a number of organisms and are involved in a variety of cellular processes. Vps1p is most similar to mammalian dynamin and the Drosophila Shibire protein, both of which have been shown to play a role in an early step of endocytosis. To identify proteins that interact with Vps1p, a genetic screen was designed to isolate multicopy suppressors of dominant-negative vps1 mutations. One such suppressor, MVP1, that exhibits genetic interaction with VPS1 and is itself required for vacuolar protein sorting has been isolated. Overproduction of Mvp1p will suppress several dominant alleles of VPS1, and suppression is dependent on the presence of wild-type Vps1p. MVP1 encodes a 59-kDa hydrophilic protein, Mvp1p, which appears to colocalize with Vps1p in vps1d and vps27 delta yeast cells. We therefore propose that Mvp1p and Vps1p act in concert to promote membrane traffic to the vacuole.
Mol Cell Biol 1995 Mar
PMID:The Saccharomyces cerevisiae MVP1 gene interacts with VPS1 and is required for vacuolar protein sorting. 786 58

It has recently been suggested that pleckstrin homology (PH) domains bind specifically to phospholipids, with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) being most strongly bound. This observation suggests that PH domains may be responsible for membrane association of proteins in which they occur. Further, this membrane association may be regulated by enzymes that modify lipid head groups to which PH domains may bind. We have studied the binding of phospholipids to the PH domain of human dynamin, a 100 kDa GTPase that is involved in the initial stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We confirm that PtdIns(4,5)P2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a specific site on the surface of dynamin PH domain of which binding of gIns(1,4,5)P3 (the head-group skeleton of PtdIns(4,5)P2) occurs. The relative affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, therefore, that the interaction of these phospholipids with dynamin is likely to occur via the PH domain. Given the fact that PH domains are often found in proteins associated with GTPase activity, or in guanine nucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis.
J Mol Biol 1996 Jan 12
PMID:Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity. 856 61

Dynamins are GTPases which support receptor-mediated endocytosis and bind to several tyrosine kinase receptor-associated proteins known to mediate cell proliferation and differentiation. We have recently established that dynamin expression correlates with normal neuronal (Torre et al., J. Biol. Chem., 269 (1994) 32411-32417) and acinar pancreatic cell differentiation (Cook et al., Mol. Biol. Cell, 6 (1995) 405a). To begin to understand the role of dynamin in neoplastic pancreatic cell differentiation, we have followed the expression of this protein by immunohistochemistry during the development of pancreatic tumors in a mancozeb-nitrosomethylurea (NMU)-based carcinogenesis model recently developed in our laboratory (Monis and Valentich, Carcinogenesis, 14 (1993) 929-933). After a single intraperitoneal injection (50 mg/g body wt) of this carcinogen, rats fed with mancozeb develop pancreatic focal acinar hyperplasia (FACH), dysplastic foci (DYF) displaying acinar-like and ductular-like structures, and ductular-like carcinoma in situ (CIS). After histochemical staining using a monoclonal anti-dynamin antibody, high levels of this protein are consistently observed in well-differentiated acinar tumors (FACH). In contrast, dynamin immunoreactivity is almost undetectable in more advanced lesions showing a ductular-like phenotype (ductular-like DYF and CIS). This change in the expression pattern of dynamin during the progression of acinar into ductular-like DYF and CIS lesions correlates with recent findings from our laboratory showing a differential expression pattern for dynamin in pancreatic cells during embryonic development, with ductular-like precursor cells expressing low levels of this protein. Based upon these results, we conclude that more advanced ductular-like neoplastic cells induced by the carcinogen NMU in rat pancreas behave phenotypically like pancreatic precursor cells in their pattern of expression for dynamin.
 ...
PMID:Expression of dynamin immunoreactivity in experimental pancreatic tumors induced in rat by mancozeb-nitrosomethylurea. 860 75

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N [1H] nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 microM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 approximately 0.9) and from the local correlation time < 100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops beta 3/beta 4 and beta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S2 approximately 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, tau loc) when protein molecular mass (tau c) increases. The use of negative values of the steady state 15N[1H] NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good-agreement with the NMR data for the amide NH groups located in the protein core.
J Mol Biol 1997 Feb 14
PMID:The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration. 905 79

Dynamin is a neuronal phosphoprotein and a GTPase enzyme which mediates late stages of endocytosis in both neural and non-neural cells. Current knowledge about dynamin is reviewed with particular emphasis on its structure and regulation with respect to phosphorylation, protein-protein interactions and phospholipid binding. The major themes are the biochemical regulation of dynamin, its effects on dynamin's GTPase activity and how this might relate to assembling the 'fission ring' that brings about vesicle retrieval. Dynamin I is an isoform of the enzyme primarily located in the central and peripheral nervous systems, where it is enriched in areas of abundant synaptic contacts. Dynamin I undergoes protein-protein interactions via its proline-rich domain at the C-terminus and these can elevate its N-terminal GTPase activity. Dynamin I interacts with multiple proteins in the nerve terminal, including SH3 domain-containing proteins such as amphiphysin and potentially with other proteins such as betagamma subunits. These regulate its role in endocytosis by targeting dynamin I to specific subcellular locations of retrieval. Dynamin I is phosphorylated in vivo by PKC and dephosphorylated on depolarization and calcium influx into nerve terminals in parallel with the coupled events of exocytosis and endocytosis. In late stages of synaptic vesicle retrieval dynamin I undergoes stimulated assembly into a collar, or fission ring, that surrounds the neck of recycling synaptic vesicles. Activation of GTP hydrolysis probably then generates the free synaptic vesicle, which can be refilled with neurotransmitters. This targeting and assembly may involve sequential steps including recruitment of AP-2 to synaptotagmin on the synaptic vesicle, and recruitment of amphiphysin, dynamin I, and synaptojanin. In addition to synaptic vesicle retrieval, dynamin has been associated with intracellular events mediated by growth factor receptors, insulin receptors and the beta-adrenergic receptor. This is likely to reflect targeting of these receptors for endocytosis soon after their activation. However, does it also suggest a broader role for dynamin in other aspects of intracellular signalling pathways?
Mol Membr Biol
PMID:Dynamin, endocytosis and intracellular signalling (review). 911 59

A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.
Mol Biol Cell 1997 Aug
PMID:Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development. 928 13

Dynamin and G-proteins both are guanosine triphosphate (GTP) binding proteins, with dynamin active in cellular membrane trafficking and G-proteins in intracellular signal transduction. Here we demonstrate that dynamin physically and functionally interacts with G-protein betagamma-subunits in neuroendocrine GH4C1 cells, on stimulation with thyrotropin-releasing hormone and somatostatin. The interaction appears to be of high affinity and inhibitory on dynamin GTPase activity, mediated by the pleckstrin homology domain and regulated both by the G-protein alpha-subunit and by guanosine nucleotides. Thus, dynamin may target particular sites for receptor-mediated endocytosis by sharing betagamma-subunits with the alpha subunit of G-proteins in neuroendocrine cells.
Mol Cell Endocrinol 1997 Sep 19
PMID:Molecular interactions between dynamin and G-protein betagamma-subunits in neuroendocrine cells. 932 47

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.
Somat Cell Mol Genet 1997 Mar
PMID:Isolation of human ear specific cDNAs and construction of cDNA libraries from surgically removed small amounts of inner ear tissues. 933 Jun 37


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