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Richard A Kahn

Publications and source records attributed to Richard A Kahn.

At least 19 recordsLinked to original sources
PubMed2006

Arl2 and Arl3 regulate different microtubule-dependent processes.

Arl2 and Arl3 are closely related members of the Arf family of regulatory GTPases that arose from a common ancestor early in eukaryotic evolution yet retain extensive structural, biochemical, and functional features. The presence of Arl3 in centrosomes, mitotic spindles, midzones, midbodies, and cilia are all supportive of roles in microtubule-dependent processes. Knockdown of Arl3 by siRNA resulted in changes in cell morphology, increased acetylation of alpha-tubulin, failure of cytokinesis, and increased number of binucleated cells. We conclude that Arl3 binds microtubules in a regulated manner to alter specific aspects of cytokinesis. In contrast, an excess of Arl2 activity, achieved by expression of the [Q70L]Arl2 mutant, caused the loss of microtubules and cell cycle arrest in M phase. Initial characterization of the underlying defects suggests a defect in the ability to polymerize tubulin in the presence of excess Arl2 activity. We also show that Arl2 is present in centrosomes and propose that its action in regulating tubulin polymerization is mediated at centrosomes. Somewhat paradoxically, no phenotypes were observed Arl2 expression was knocked down or Arl3 activity was increased in HeLa cells. We conclude that Arl2 and Arl3 have related but distinct roles at centrosomes and in regulating microtubule-dependent processes.

ADP-Ribosylation Factors↗
PubMed2006

Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins.

The Ras superfamily is comprised of at least four large families of regulatory guanosine triphosphate-binding proteins, including the Arfs. The Arf family includes three different groups of proteins: the Arfs, Arf-like (Arls), and SARs. Several Arf family members have been very highly conserved throughout eukaryotic evolution and have orthologues in evolutionally diverse species. The different means by which Arf family members have been identified have resulted in an inconsistent and confusing array of names. This confusion is further compounded by differences in nomenclature between different species. We propose a more consistent nomenclature for the human members of the Arf family that may also serve as a guide for nomenclature in other species.

ADP-Ribosylation Factors↗
PubMed2005

Isoform-selective effects of the depletion of ADP-ribosylation factors 1-5 on membrane traffic.

The ADP-ribosylation factors (Arfs) are six proteins within the larger Arf family and Ras superfamily that regulate membrane traffic. Arfs all share numerous biochemical activities and have very similar specific activities. The use of dominant mutants and brefeldin A has been important to the discovery of the cellular functions of Arfs but lack specificity between Arf isoforms. We developed small interference RNA constructs capable of specific depletion of each of the cytoplasmic human Arfs to examine the specificity of Arfs in live cells. No single Arf was required for any step of membrane traffic examined in HeLa cells. However, every combination of the double knockdowns of Arf1, Arf3, Arf4, and Arf5 yielded a distinct pattern of defects in secretory and endocytic traffic, demonstrating clear specificity for Arfs at multiple steps. These results suggest that the cooperation of two Arfs at the same site may be a general feature of Arf signaling and provide candidates at several cellular locations that when paired with data on the localization of the many different Arf guanine nucleotide exchange factors, Arf GTPase activating proteins, and effectors will aid in the description of the mechanisms of specificity in this highly conserved and primordial family of regulatory GTPases.

ADP-Ribosylation Factors↗
PubMed2005

Assays used in the analysis of Arl2 and its binding partners.

Arl2 is a approximately 20 kDa GTPase in the ADP-ribosylation factor (Arf) family within the Ras superfamily with roles in microtubule dynamics that impact the cytoskeleton, cell division, and cytokinesis. Arl2 has been implicated as a regulator of the pathway responsible for formation of properly folded tubulin heterodimers and in adenine nucleotide transport in mitochondria. The identification and characterization of Arl2 binding partners and regulators of Arl2 activities are critical steps in the further dissection of these and likely other Arl2-dependent functions. This chapter describes methods for preparing recombinant Arl2, loading different radiolabeled guanine nucleotides onto the GTPase, identifying high-affinity Arl2 binding proteins, and assaying Arl2 GTPase activating proteins (GAPs). These methods may also prove useful for studies of other Arls or other GTPases.

Animals↗
PubMed2004

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells.

The Legionella pneumophila Dot/Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot/Icm system. In this study, the mechanism of Dot/Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot/Icm system. A leucine (L372) residue at the -3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot/Icm system, most other Dot/Icm substrates were found to have amino acid residues with similar physical properties at their -3 or -4 C-terminal positions. These data demonstrate that the Dot/Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides.

Adenylate Cyclase Toxin↗
PubMed2004

Structural perturbations in human ADP ribosylation factor-1 accompanying the binding of phosphatidylinositides.

ADP ribosylation factors (Arfs) are members of a family of Ras-related GTPases that regulate a wide variety of intracellular signaling pathways, including the regulation of membrane traffic and organelle morphology. Arfs perform these functions through interactions with Arf-specific guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and effectors. Signaling phosphatidylinositides, most commonly phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) or phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)), have been shown previously to regulate the activities of a number of these regulators and effectors of Arf. The ability of Arf itself to bind these same phosphatidylinositides also has been reported previously, though without much structural detail. We investigated the ability of human Arf1.GDP (Arf1.GDP) to bind myo-inositol (1,4,5)-trisphosphate (I(1,4,5)P(3)), the soluble headgroup for PI(4,5)P(2), and a short acyl-chain soluble PI(4,5)P(2) analogue using heteronuclear single quantum coherence (HSQC)-based NMR techniques. A patch of positive electrostatic potential on the surface of Arf1.GDP is identified as being directly involved in ligand binding, but structural and stability changes extending to the N-terminal helix and nucleotide-binding site of Arf1 are also documented. The identified binding site and the resultant structural changes are discussed in terms of a possible influence of phosphatidylinositides on the binding of Arf1 to Arf1-GEF and subsequent nucleotide release.

ADP-Ribosylation Factor 1↗
PubMed2004

The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site.

The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the "glutamic finger," conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.

ADP-Ribosylation Factors↗
PubMed2004

Conformational changes in human Arf1 on nucleotide exchange and deletion of membrane-binding elements.

Conformational changes associated with nucleotide exchange or truncation of the N-terminal alpha-helix of human Arf1 have been investigated by using forms of easily acquired NMR data, including residual dipolar couplings and amide proton exchange rates. ADP-ribosylation factors (Arfs) are 21-kDa GTPases that regulate aspects of membrane traffic in all eukaryotic cells. An essential component of the biological actions of Arfs is their ability to reversibly bind to membranes, a process that involves exposure of the myristoylated N-terminal amphipathic alpha-helix upon activation and GTP binding. Deletion of this helix results in a protein, termed Delta17Arf1, that has a reduced affinity for GDP and the ability to bind GTP in the absence of lipids or detergents. Previous studies, comparing crystal structures for Arf1.GDP and Delta17Arf1.GTP, identified several regions of structural variation and suggested that these be associated with nucleotide exchange rather than removal of the N-terminal helix. However, separation of conformational changes because of nucleotide binding and N-terminal truncation cannot be addressed in comparing these structures, because both the bound nucleotide and the N terminus differ. Resolving the two effects is important as any structural changes involving the N terminus may represent membrane-mediated conformational adjustments that precede GTP binding. Results from NMR experiments presented here on Arf1.GDP and Delta17Arf1.GDP in solution reveal substantial structural differences that can only be associated with N-terminal truncation.

ADP-Ribosylation Factor 1↗
PubMed2004

Cyclic enterobacterial common antigen: potential contaminant of bacterially expressed protein preparations.

We have previously reported the identification of the cyclic enterobacterial common antigen (ECA(CYC)) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N -acetyl signals of ECA(CYC) have been observed in (15)N-(1)H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECA(CYC) in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate.

Antigens, Bacterial↗
PubMed2004

Functional genomic analysis of the ADP-ribosylation factor family of GTPases: phylogeny among diverse eukaryotes and function in C. elegans.

ADP-ribosylation factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The approximately 100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). Injection of double-stranded RNA (dsRNA) encoding Arf-1 or Arf-3 into N2 hermaphrodites produced embryonic lethality in their offspring and, later, sterility in the injected animals themselves. Injection of Arl-2 dsRNA resulted in a disorganized germline and sterility in early offspring, with later offspring exhibiting an early embryonic arrest. Thus, of the six Arf family members examined in C. elegans, at least three are required for embryogenesis. These data represent the first analysis of the role(s) of multiple members of this family in the development of a multicellular organism.

ADP-Ribosylation Factors↗
PubMed2004

Multiple phosphorylation events regulate the subcellular localization of GGA1.

GGAs comprise a family of Arf-dependent coat proteins or adaptors that regulate vesicle traffic from the trans-Golgi network (TGN). GGAs bind activated Arf, cargo, and additional components necessary for vesicle budding through interactions with their four functional domains: VHS, GAT, hinge, and GAE. We identified three sites of phosphorylation in GGA1 by tandem mass spectrometry: S268 and T270 in the GAT domain and S480 in the hinge. Expression of HA-GGA1 in mammalian cells and comparison to endogenous GGA1 confirmed their localization to late Golgi compartments. In contrast, mutations that mimic the phosphoprotein (HA-GGA1[S268D] or HA-GGA1[T270D]) at either of the sites in the GAT domain caused a decrease in the colocalization with markers of the Golgi and TGN and an increase in puncta in cytoplasm. Quantitative comparisons of the extent of colocalization of GGA1 proteins with the known components of GGA1 vesicles revealed that the composition of those markers tested in HA-GGA1[S268D] and HA-GGA1[T270D] vesicles were indistinguishable from those of HA-GGA1 vesicles. We conclude that phosphorylation of the GAT domain can stabilize the coat proteins bound and thus regulate the rate of coat protein dissociation.

ADP-Ribosylation Factors↗
PubMed2004

Reverse two-hybrid techniques in the yeast Saccharomyces cerevisiae.

Use of the yeast two-hybrid system has provided definition to many previously uncharacterized pathways through the identification and characterization of novel protein-protein interactions. The two-hybrid system uses the bi-functional nature of transcription factors, such as the yeast enhancer Gal4, to allow protein-protein interactions to be monitored through changes in transcription of reporter genes. Once a positive interaction has been identified, either of the interacting proteins can mutate, either by site-specific or randomly introduced changes, to produce proteins with a decreased ability to interact. Mutants generated using this strategy are very powerful reagents in tests of the biological significance of the interaction and in defining the residues involved in the interaction. Such techniques are termed reverse two-hybrid methods. We describe a reverse two-hybrid method that generates loss-of-interaction mutations of the catalytic subunit of the Escherichia coli heat-labile toxin (LTA1) with decreased binding to the active (GTP-bound) form of human ARF3, its protein cofactor.

ADP-Ribosylation Factors↗
PubMed2003

Cytosolic Arl2 is complexed with cofactor D and protein phosphatase 2A.

Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because approximately 90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of approximately 300 kDa. This complex was purified approximately 500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis.

Animals↗
PubMed2003

Munc18 interacting proteins: ADP-ribosylation factor-dependent coat proteins that regulate the traffic of beta-Alzheimer's precursor protein.

Coat proteins cycle between soluble and membrane-bound locations at the time of vesicle biogenesis and act to regulate the assembly of the vesicle coat that determines the specificity in cargo selection and the destination of the vesicle. A transmembrane cargo protein, an Arf GTPase, and a coat protein (e.g. COPs, APs, or GGAs) are minimal components required for budding of vesicles. Munc18 interacting proteins (MINTs) are a family of three proteins implicated in the localization of receptors to the plasma membrane. We show that MINTs bind Arfs directly, co-localize with Arf and the Alzheimer's precursor protein (beta-APP) to regions of the Golgi/trans-Golgi network, and can co-immunoprecipitate clathrin. We demonstrate that MINTs bind Arfs through a region of the PTB domain and the PDZ2 domain, and Arf-MINT interaction is necessary for the increased cellular levels of beta-APP produced by MINT overexpression. Knockdown (small interference RNA) experiments implicate beta-APP as a transmembrane cargo protein that works together with MINTs. We propose that MINTs are a family of Arf-dependent, vesicle-coat proteins that can regulate the traffic of beta-APP.

ADP-Ribosylation Factors↗
PubMed2003

Four ARF GAPs in Saccharomyces cerevisiae have both overlapping and distinct functions.

Previous studies in yeast have revealed the presence of four proteins with a conserved, cysteine-rich, ARF GAP domain that share the ability to suppress the conditional growth defect of the arf1-3 mutant. Three of these proteins have been shown previously to be ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs). We now demonstrate that the fourth also exhibits in vitro ARF GAP activity and correlates the suppressor and ARF GAP activities for all four. Because the four ARF GAP proteins are quite diverse outside the ARF GAP domain, a genetic analysis was undertaken to define the level of functional cross-talk between them. A large number of synthetic defects were observed that point to a high degree of functional overlap among the four ARF GAPs. However, several differences were also noted in the ability of each gene to suppress the synthetic defects of others and in the impact of single or combined deletions on assays of membrane traffic. We interpret these results as supportive evidence for roles of ARF GAPs in a number of distinct, essential cellular processes that include cell growth, protein secretion, endocytosis and cell cycling. The description of the specificities of the ARF GAPs for the different responses is viewed as a necessary first step in dissecting biologically relevant pathways through a functionally overlapping family of signalling proteins.

ADP-Ribosylation Factors↗
PubMed2002

ARL1 and membrane traffic in Saccharomyces cerevisiae.

To examine the functions of the Arf-like protein, Arl1p, in Saccharomyces cerevisiae, a null allele, arl1delta::HIS3, was constructed in two strains. In one background only, loss of ARL1 resulted in temperature-sensitive (ts) growth (suppressed on high-osmolarity media). Allelic variation at the SSD1 locus accounted for differences between strains. Strains lacking ARL1 exhibited several defects in membrane traffic. First, arl1delta strains secreted less protein as measured by TCA-precipitable radioactivity found in the media of [(35)S]-labelled cells. A portion of newly synthesized carboxypeptidase Y (CPY) was secreted rather than correctly targeted to the vacuole. Uptake of the fluid-phase marker, lucifer yellow, was reduced. All these phenotypes were exacerbated in an ssd1 background. The ts phenotype of the arl1deltassd1 strain was suppressed by YPT1, the yeast Rab1a homologue, suggesting that ARL1 and YPT1 have partially overlapping functions. These findings demonstrate that ARL1 encodes a regulator of membrane traffic.

ADP-Ribosylation Factors↗