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PubMed2006

Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved.

The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.

Animals↗
PubMed2003

Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway.

Cytosolic internalization is a requirement for the toxicity of secretory ribonucleases. Here, we investigate the mechanism of internalization of Onconase (ONC), a toxic protein, and ribonuclease A (RNase A), a nontoxic homolog. Microscopy studies indicate that both ribonucleases readily bind to the cell surface and are internalized via acidic vesicles. Blocking dynamin-dependent endocytosis prevents transferrin internalization but does not hinder RNase A internalization. ONC and G88R RNase A, which is a toxic variant, demonstrate enhanced cytotoxicity in the absence of clathrin- and dynamin-mediated endocytosis. The cytosolic entry of ribonucleases does not require an acidic environment or transport to the ER and probably occurs from endosomes. Thus, common proteins - secretory ribonucleases - enter the cytosol by a pathway that is distinct from that of other known toxins.

Bodily Secretions↗
PubMed2006

Dynamin as a mover and pincher during cell migration and invasion.

The large GTPase dynamin, long known for its role in endocytosis, has most recently been implicated as a facilitator of cell migration and invasion. Recent observations link dynamin to the cycle of membrane expansion and retraction essential for cell motility. Its role in actin polymerization, membrane deformation and vesiculation, and focal adhesion dynamics are all important for this process, and the new findings provide exciting directions for studies of this ubiquitous and diverse protein family.

Actins↗
PubMed2004

The role of dynamin in the assembly and function of podosomes and invadopodia.

Cells make contact with the extracellular matrix (ECM) through extensions of the plasma membrane; these range from irregular dynamic structures, e.g. lamellipodia, ruffles and pseudopodia, to more localized and highly defined protrusions, e.g. podosomes and invadopodia. Both might be instruments through which cells sample the immediate extracellular environment and maintain polarized activities such as chemotaxis and focal degradation of the matrix. Podosomes are expressed in cells of the monocytic lineage, and most studies point to a role for podosomes in adhesion/motility. Invadopodia are prominent in certain aggressive cancer cells (or transformed cells) and appear to be directly responsible for focal ECM degradation. Recent studies have revived interest in these structures in terms of the actin regulation machinery. Within this framework, the atypical GTP-binding protein dynamin, a central modulator of protrusive events, has been associated to podosome and invadopodia structure and function. Here, we specifically discuss the role played by dynamin in controlling the activities and function of these structures.

Animals↗
PubMed2003

Clathrin- and dynamin-dependent coated vesicle formation from isolated plasma membranes.

We have developed a new rapid cell-free assay for endocytic clathrin-coated vesicle formation using highly purified rat liver plasma membrane sheets. After incubation in the presence of cytosol and nucleotides, released vesicles were collected by high-speed centrifugation and incorporated cargo receptors were detected by Western blotting. Three different cargo receptors were internalized into vesicles while a receptor, known to be excluded from coated pits, was not. The recruitment of cargo receptors into the vesicle fraction was cytosol, ATP and temperature-dependent and was enhanced by addition of GTP. Vesicle formation in this assay was confirmed by subcellular fractionation and EM analysis. Plasma membranes stripped of their endogenous coat proteins with 0.5 m Tris retained vesicle formation activity, which was highly dependent on clathrin and dynamin. Coat proteins and dynamin were not sufficient for clathrin-coated vesicle formation, and other peripheral membrane proteins recruited from the cytosol are required. The nonhydrolyzable ATP analogue, AMPPNP did not support clathrin-coated vesicle formation; however, surprisingly, GTP gamma S was as effective as GTP. This assay will provide a powerful tool to dissect the minimum machinery and to probe the hierarchy of events involved in cargo selection and endocytic clathrin-coated vesicle formation.

Adaptor Protein Complex 2↗
PubMed2003

[Molecular cloning of Plasmodium yoelii dynamin-like protein (PyDyn) gene and the immunological character of its domains].

OBJECTIVE: To identify and clone a new full ORF gene of PyDyn (Plasmodium yoelii dynamin-like protein), and examine the protection of their expression products. METHOD: Using the P. yoelii Genome technology and RT-PCR. RESULTS: The full ORF gene of PyDyn was amplified from mRNA of the erythrocytic stage of P. yoelii., three domains of PyDyn were expressed in E. coli., and the fairly positive immunogenicity of them was showed by IFA. The full ORF gene of PyDyn was 2,433 bp and encode 811 amino acids. Its Gene Bank access number is AF458071. PyDyn belongs to the dynamin-like protein family according to its property. CONCLUSION: The new full ORF gene of PyDyn is obtained and identified; their expressed domains are probably new candidates for malaria vaccine.

Amino Acid Sequence↗
PubMed1993

The GTPase dynamin binds to and is activated by a subset of SH3 domains.

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.

Amino Acid Sequence↗
PubMed2005

Assay and properties of the mitochondrial dynamin related protein Opa1.

Opa1, also known as Mgm1 in yeast, is a mitochondrial member of the dynamin family. Unlike other dynamin family members, Opa1 has an N-terminal mitochondrial targeting sequence, suggesting that this protein is imported into mitochondria. Here, we describe biochemical techniques, such as mitochondrial isolation, digitonin extraction, a protease protection assay, and carbonate extraction, that were used to determine that mammalian Opa1 resides in the intermembrane space where it is tightly bound to the inner membrane. In addition, we describe bacterial expression of the Opa1 GTPase domain, methods for purification, and an in vitro assay for GTP hydrolysis.

Animals↗
PubMed2004

Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynamin-like GTPase MxB.

Mx proteins form a subfamily of the dynamin-like GTPases, which have well established roles in cellular trafficking. Some Mx proteins (e.g., human MxA) have antiviral activity and are tightly regulated by type I IFNs. Others (e.g., human MxB) lack antiviral activity and are thought to have normal cellular functions that remain undefined. Consistent with this hypothesis, we report that MxB is expressed without IFN treatment. MxB seems to be exclusively extranuclear and is concentrated at the cytoplasmic face of nuclear pores, suggesting a role in their regulation. We find that expression of dominant negative (GTPase-defective) MxB mutants efficiently blocks nuclear import and causes a delay in G(1)/S cell-cycle progression. MxB depletion using RNA interference (RNAi) leads to a similar cell-cycle defect but does not block nuclear import. MxB therefore seems not to be required for nuclear import per se but may instead regulate its efficiency and/or kinetics. These studies indicate an unexpected role for a dynamin-like protein in nucleocytoplasmic trafficking and suggest that a related function might be usurped by its antiviral relatives.

Active Transport, Cell Nucleus↗
PubMed2003

Calcium-sensing receptor-mediated ERK1/2 activation requires Galphai2 coupling and dynamin-independent receptor internalization.

The calcium-sensing receptor (CaR) recently has been shown to activate MAP kinase (ERK1/2) in various cell types as well as in heterologous expression systems. In this study we show that the CaR agonist NPS R-467 (1 microm), which does not activate the CaR by itself, robustly activates ERK1/2 in the presence of a low concentration of Ca(2+) (0.5 mm CaCl(2)) in human embryonic kidney (HEK) cells permanently expressing the human CaR (HEK-hCaR). Ca(2+) (4 mm) also activates ERK1/2 but with differing kinetics. CaR-dependent ERK1/2 activation begins to desensitize to 4 mm Ca(2+) after 10 min, whereas there is no desensitization to NPS R-467/CaCl(2) as late as 4 h. Moreover, recovery from desensitization occurs as rapidly as 30 min with 4 mm CaCl(2). Pretreatment of HEK-hCaR cells with concanavalin A (250 microg/ml) to block CaR internalization completely eliminated the NPS R-467/CaCl(2)-mediated ERK1/2 activation but did not block the 2-min time point of 4 mm Ca(2+)-mediated ERK1/2 activation. Neither dominant-negative dynamin (K44A) nor dominant-negative beta-arrestin inhibited ERK1/2 activation by either CaR agonist treatment, suggesting that CaR-elicited ERK1/2 signaling occurs via a dynamin-independent pathway. Pertussis toxin pretreatment partially attenuated the 4 mm Ca(2+)-ERK1/2 activation; this attenuated activity was completely restored by co-expression of the Galpha(i2) (C351I) but not Galpha(i1) (C351I) or Galpha(i3) (C351I) G proteins, PTX-insensitive G protein mutants. Taken together, these data suggest that both 4 mm Ca(2+) and NPS R-467/CaCl(2) activate ERK1/2 via distinguishable pathways in HEK-hCaR cells and may represent a nexus to differentially regulate differentiation versus proliferation via CaR activation.

Arrestins↗
PubMed2003

A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation.

Rapidly reversible, temperature-sensitive (ts) paralytic mutants of Drosophila have been useful in delineating immediate in vivo functions of molecules involved in synaptic transmission. Here we report isolation and characterization of orangi (org), an enhancer of shibire (shi), a ts paralytic mutant in Drosophila dynamin. org is an allele of the stress sensitive B (sesB) locus that encodes a mitochondrial adenine nucleotide translocase (ANT) and results in a unique ts paralytic behavior that is accompanied by a complete loss of synaptic transmission in the visual system. sesB(org) reduces the restrictive temperature for all shi(ts) alleles tested except for shi(ts1). This characteristic allele-specific interaction of sesB(org) with shi is shared by abnormal wing discs (awd), a gene encoding nucleoside diphosphate kinase (NDK). sesB(org) shows independent synergistic interactions, an observation that is consistent with a shared pathway by which org and awd influence shi function. Genetic and electrophysiological analyses presented here, together with the observation that the sesB(org) mutation reduces biochemically assayed ANT activity, suggest a model in which a continuous mitochondrial ANT-dependent supply of ATP is required to sustain NDK-dependent activation of presynaptic dynamin during a normal range of synaptic activity.

Alleles↗
PubMed2004

An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division.

Peroxisomes undergo dramatic changes in size, shape, number, and position within the cell, but the division process of peroxisomes has not been characterized. We screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants). In one of these mutants, apm1, the peroxisomes are long and reduced in number, apparently as a result of inhibition of division. We showed that APM1 encodes dynamin-related protein 3A (DRP3A), and that mutations in APM1/DRP3A also caused aberrant morphology of mitochondria. The transient expression analysis showed that DRP3A is associated with the cytosolic side of peroxisomes. These findings indicate that the same dynamin molecule is involved in peroxisomal and mitochondrial division in higher plants. We also report that the growth of Arabidopsis, which requires the cooperation of various organelles, including peroxisomes and mitochondria, is repressed in apm1, indicating that the changes of morphology of peroxisomes and mitochondria reduce the efficiency of metabolism in these organelles.

Amino Acid Sequence↗
PubMed1995

Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family.

Dynamin (Dyn) is a member of a novel group of GTPases which was initially identified as a microtubule-binding protein with a role in vectorial movement. Three distinct Dyn-encoding genes (DYN I, II and III), with a neuronal-, ubiquitous or testis-specific expression, respectively, have been identified in rat. In man, only DYN I has so far been characterized. We have previously isolated a genomic DNA fragment implicated in the correction of mitomycin C hypersensitivity of cells from a Fanconi anemia patient belonging to genetic complementation group D (FA(D)). Using this probe, we have cloned a human complementary DNA designated hDYN II encoding a ubiquitous Dyn isoform. The predicted protein consists of 866 amino acids (97.5 kDa). Dyn proteins exhibit a high degree of evolutionary conservation: hDyn II is 98% identical to rat Dyn II and 73% identical to hDyn I. A unique 3.6-kb transcript is found in all human tissues examined and it is more abundant in skeletal muscle and heart. This transcript is also expressed in tissue-culture cells. The hDYN II message is present and not mutated in the FA(D) patient studied. In addition to the GTP-binding domain and motifs associated with regulatory function, the hDyn II protein contains a noticeable number of concensus motifs for p34Cdc2 kinase phosphorylation which may indicate a potential role at the G2/mitosis transition. The sequence reported here should allow a more complete analysis of Dyn function(s) in man.

Amino Acid Sequence↗
PubMed2005

A continuous, regenerative coupled GTPase assay for dynamin-related proteins.

Dynamin-related proteins (DRPs) compose a diverse family of proteins that function, through GTPase stimulated self-assembly, to remodel cellular membranes. The molecular mechanism by which DRPs mediate membrane remodeling events and the specific role of their GTPase cycle is still not fully understood. Although DRPs are members of the GTPase superfamily, they possess unique kinetic properties. In particular, they have relatively low affinity for guanine nucleotides and, under conditions that favor self-assembly, they have high rates of GTP turnover. Established fixed time point assays used for the analysis of assembly stimulated GTPase activity are prone to inaccuracies due to substrate depletion and are also limited by lack of time resolution. We describe a simple, continuous, coupled GTP regenerating assay that tackles the limitations of the fixed time point assays and can be used for the kinetic analysis of DRP GTP hydrolysis under unassembled and assembled conditions.

Dynamin I↗
PubMed1990

Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins.

A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.

Amino Acid Sequence↗
PubMed2002

Inositol hexakisphosphate promotes dynamin I- mediated endocytosis.

Membrane homeostasis is maintained by exocytosis and endocytosis. The molecular mechanisms regulating the interplay between these two processes are not clear. Inositol hexakisphosphate (InsP(6)) is under metabolic control and serves as a signal in the pancreatic beta cell stimulus-secretion coupling by increasing Ca(2+)-channel activity and insulin exocytosis. We now show that InsP(6) also promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This effect of InsP(6) depends on calcineurin-induced dephosphorylation and is accounted for by both activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin and thereby formation of phosphatidylinositol 4,5-bisphosphate. In regulating both exocytosis and endocytosis, InsP(6) thus may have an essential integral role in membrane trafficking.

Animals↗
PubMed1997

Delta and kappa opioid receptors are differentially regulated by dynamin-dependent endocytosis when activated by the same alkaloid agonist.

Many alkaloid drugs used as analgesics activate multiple opioid receptors. Mechanisms that distinguish the actions of these drugs on the regulation of individual micro, delta, and kappa receptors are not understood. We have observed that individual cloned opioid receptors differ significantly in their regulation by rapid endocytosis in the presence of alkaloid drug etorphine, a potent agonist of mu, delta, and kappa opioid receptors. Internalization of epitope-tagged delta opioid receptors from the plasma membrane is detectable within 10 min in the presence of etorphine. In contrast, kappa receptors expressed in the same cells remain in the plasma membrane and are not internalized for >/=60 min, even when cells are exposed to saturating concentrations of etorphine. The rapid internalization of delta receptors is specifically inhibited in cells expressing K44E mutant dynamin I, suggesting that type-specific internalization of opioid receptors is mediated by clathrin-coated pits. Examination of a series of chimeric mutant kappa/delta receptors indicates that at least two receptor domains, including the highly divergent carboxyl-terminal cytoplasmic tail, determine the type specificity of this endocytic mechanism. We conclude that structurally homologous opioid receptors are differentially sorted by clathrin-mediated endocytosis following activation by the same agonist ligand. These studies identify a fundamental mechanism of receptor regulation mediating type-specific effects of analgesic drugs that activate more than one type of opioid receptor.

Adenylyl Cyclases↗
PubMed2004

Intra- and intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1.

Mammalian Drp1 is a dynamin-like GTPase required for mitochondrial fission. Although it exists primarily as a cytosolic homo-tetramer in vivo, it can also self-assemble into higher order structures on the mitochondrial outer membrane, where it is required for proper mitochondrial division. Functional studies and sequence comparisons have revealed four different structural domains in Drp1, comprising N-terminal GTP-binding, middle, insert B, and C-terminal GTPase effector (GED) domains. Here we describe an intramolecular interaction within Drp1 between the GED and the N-terminal GTP-binding and middle domains. A point mutation (K679A) within the C-terminal GED domain inhibits this intramolecular association, without affecting the formation of Drp1 tetramers or the intermolecular associations among isolated C-terminal domains. Mutant Drp1 K679A exhibits impaired GTPase activity, and when overexpressed in mammalian cells it decreases mitochondrial division. Sedimentation experiments indicate that the K679A mutation either increases Drp1 complex formation or, more likely, decreases complex disassembly as compared with wild-type Drp1. Taken together, these data suggest that the C-terminal GED domain is important for stimulation of GTPase activity, formation and stability of higher order complexes, and efficient mitochondrial division.

Amino Acid Sequence↗